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    Structured Review

    Thermo Fisher total rna
    Gene expression profile in <t>IVD</t> cells treated with fHAs . Human IVD cells were incubated for 18 hours with hyaluronic acid fragments (fHAs) at either 5 or 20 μg/ml ( n = 4 to 6) and <t>RNA</t> harvested for analysis by qRT-PCR. Values were normalized to TATA-Box binding protein ( TBP ) mRNA and expressed as 2 -Δ C T . Statistical analysis was performed using the Kruskal-Wallis one-way analysis of variance for multiple group comparisons followed by the Mann-Whitney U test for comparisons between two groups, * P
    Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 277 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Hyaluronic acid fragments enhance the inflammatory and catabolic response in human intervertebral disc cells through modulation of toll-like receptor 2 signalling pathways"

    Article Title: Hyaluronic acid fragments enhance the inflammatory and catabolic response in human intervertebral disc cells through modulation of toll-like receptor 2 signalling pathways

    Journal: Arthritis Research & Therapy

    doi: 10.1186/ar4274

    Gene expression profile in IVD cells treated with fHAs . Human IVD cells were incubated for 18 hours with hyaluronic acid fragments (fHAs) at either 5 or 20 μg/ml ( n = 4 to 6) and RNA harvested for analysis by qRT-PCR. Values were normalized to TATA-Box binding protein ( TBP ) mRNA and expressed as 2 -Δ C T . Statistical analysis was performed using the Kruskal-Wallis one-way analysis of variance for multiple group comparisons followed by the Mann-Whitney U test for comparisons between two groups, * P
    Figure Legend Snippet: Gene expression profile in IVD cells treated with fHAs . Human IVD cells were incubated for 18 hours with hyaluronic acid fragments (fHAs) at either 5 or 20 μg/ml ( n = 4 to 6) and RNA harvested for analysis by qRT-PCR. Values were normalized to TATA-Box binding protein ( TBP ) mRNA and expressed as 2 -Δ C T . Statistical analysis was performed using the Kruskal-Wallis one-way analysis of variance for multiple group comparisons followed by the Mann-Whitney U test for comparisons between two groups, * P

    Techniques Used: Expressing, Incubation, Quantitative RT-PCR, Binding Assay, MANN-WHITNEY

    2) Product Images from "TBP2 is essential for germ cell development by regulating transcription and chromatin condensation in the oocyte"

    Article Title: TBP2 is essential for germ cell development by regulating transcription and chromatin condensation in the oocyte

    Journal: Genes & Development

    doi: 10.1101/gad.535209

    TBP2 ablation results in altered oocyte-specific transcriptional profile in 2-wk-old ovaries. ( A ) Distribution of the number of probes on the microarray with respect to their FDR and fold change in 2-wk-old the Tbp2 −/− ovaries compared with the control. ( Top panel) In the histogram, bars of different colors represent the number of probes differentially hybridized at the indicated FDR threshold. ( Bottom panel) The exact number of probes is displayed in the table. At FDR = 0.12 (yellow bars), there are as many probes in the down-regulated class (negative fold change) as there are in the up-regulated one (positive fold change). ( B ) Microarray validation in 2-wk-old Tbp2 −/− and Tbp2 +/+ ovaries. Up-regulated and down-regulated genes with different FDR and fold change values (ranging from −0.22-fold to 2.5-fold) were randomly selected and their mRNA levels were analyzed by RT-qPCR. mRNA levels are shown relative to 18S RNA and are expressed relative to the control, for which mRNA levels were set at 1 (black line). Microarray (black bars) and RT-qPCR (gray bars) fold changes are average of three independent biological replicates, respectively. Error bars are SD. ( C ) Most enriched functional categories among the down-regulated and up-regulated genes according to Ingenuity Pathways. ( D ) Genes down-regulated upon TBP2 loss are enriched in oocyte-specific genes. Comparison of the up-regulated and down-regulated genes with an FDR of 0.12 with genes that are specifically expressed in the oocyte. P values for the intersection of the up-regulated and down-regulated genes are shown at the bottom . Note that only the intersection of down-regulated genes is statistically significant. ( E ) Sequence composition of the promoters belonging to the two classes of genes according to their nucleotide content. The percentage of AT content of the up-regulated (red) and down-regulated (green) gene promoters within a region from −100 to +100 bp around TSS is shown. The average AT content for the two promoter populations is also indicated. Comparison of the two groups of genes using a Student's t -test indicates that the two populations of promoters differ significantly. The TSS positions follow the current NCBI annotation.
    Figure Legend Snippet: TBP2 ablation results in altered oocyte-specific transcriptional profile in 2-wk-old ovaries. ( A ) Distribution of the number of probes on the microarray with respect to their FDR and fold change in 2-wk-old the Tbp2 −/− ovaries compared with the control. ( Top panel) In the histogram, bars of different colors represent the number of probes differentially hybridized at the indicated FDR threshold. ( Bottom panel) The exact number of probes is displayed in the table. At FDR = 0.12 (yellow bars), there are as many probes in the down-regulated class (negative fold change) as there are in the up-regulated one (positive fold change). ( B ) Microarray validation in 2-wk-old Tbp2 −/− and Tbp2 +/+ ovaries. Up-regulated and down-regulated genes with different FDR and fold change values (ranging from −0.22-fold to 2.5-fold) were randomly selected and their mRNA levels were analyzed by RT-qPCR. mRNA levels are shown relative to 18S RNA and are expressed relative to the control, for which mRNA levels were set at 1 (black line). Microarray (black bars) and RT-qPCR (gray bars) fold changes are average of three independent biological replicates, respectively. Error bars are SD. ( C ) Most enriched functional categories among the down-regulated and up-regulated genes according to Ingenuity Pathways. ( D ) Genes down-regulated upon TBP2 loss are enriched in oocyte-specific genes. Comparison of the up-regulated and down-regulated genes with an FDR of 0.12 with genes that are specifically expressed in the oocyte. P values for the intersection of the up-regulated and down-regulated genes are shown at the bottom . Note that only the intersection of down-regulated genes is statistically significant. ( E ) Sequence composition of the promoters belonging to the two classes of genes according to their nucleotide content. The percentage of AT content of the up-regulated (red) and down-regulated (green) gene promoters within a region from −100 to +100 bp around TSS is shown. The average AT content for the two promoter populations is also indicated. Comparison of the two groups of genes using a Student's t -test indicates that the two populations of promoters differ significantly. The TSS positions follow the current NCBI annotation.

    Techniques Used: Microarray, Quantitative RT-PCR, Functional Assay, Sequencing

    RNA Pol II activity and chromatin structure are altered in oocytes lacking TBP2. ( A , B ) Immunofluorescence analysis of control and Tbp2 −/− ovarian sections from 6-wk-old females using antibodies recognizing the Ser2-phophorylated CTD of Pol II ( A ) or all forms of the CTD of Pol II ( B ). Pol II staining is shown in red. DNA (blue) was counterstained with DAPI. Follicular stages are indicated. ( A ) The arrow points to the follicle at the stage indicated at the top . Bars: for primordial stages, 10 μm; for later stages, 50 μm. ( B ) Bar, 50 μm. ( C ) Tbp2 −/− oocytes show reduced H3K4me3 levels from the primary follicular stage. Immunostaining analysis for H3K4me3 in oocytes at different stages of follicular development from 6-wk-old females. DNA (blue) was stained with DAPI. Bar, 10 μm. Confocal acquisition was done using identical parameters to allow comparison. Hence, H3K4me3 levels in preovulatory stage oocyte in the control appear saturated. Note that Tbp2 −/− oocytes degenerate after the secondary follicular stage, as seen from the presence of follicular cells within the follicle. The dashed white line demarcates the oocyte membrane. ( D ) Altered chromatin configuration in Tbp2 −/− oocytes. Sections from control and Tbp2 −/− mice were stained with DAPI and analyzed under confocal microscopy. (NSN) Nonsurrounded nucleolus; (PSN) partially surrounded nucleolus; (SN) surrounded nucleolus. Representative sections are shown. (ND) Not determined.
    Figure Legend Snippet: RNA Pol II activity and chromatin structure are altered in oocytes lacking TBP2. ( A , B ) Immunofluorescence analysis of control and Tbp2 −/− ovarian sections from 6-wk-old females using antibodies recognizing the Ser2-phophorylated CTD of Pol II ( A ) or all forms of the CTD of Pol II ( B ). Pol II staining is shown in red. DNA (blue) was counterstained with DAPI. Follicular stages are indicated. ( A ) The arrow points to the follicle at the stage indicated at the top . Bars: for primordial stages, 10 μm; for later stages, 50 μm. ( B ) Bar, 50 μm. ( C ) Tbp2 −/− oocytes show reduced H3K4me3 levels from the primary follicular stage. Immunostaining analysis for H3K4me3 in oocytes at different stages of follicular development from 6-wk-old females. DNA (blue) was stained with DAPI. Bar, 10 μm. Confocal acquisition was done using identical parameters to allow comparison. Hence, H3K4me3 levels in preovulatory stage oocyte in the control appear saturated. Note that Tbp2 −/− oocytes degenerate after the secondary follicular stage, as seen from the presence of follicular cells within the follicle. The dashed white line demarcates the oocyte membrane. ( D ) Altered chromatin configuration in Tbp2 −/− oocytes. Sections from control and Tbp2 −/− mice were stained with DAPI and analyzed under confocal microscopy. (NSN) Nonsurrounded nucleolus; (PSN) partially surrounded nucleolus; (SN) surrounded nucleolus. Representative sections are shown. (ND) Not determined.

    Techniques Used: Activity Assay, Immunofluorescence, Staining, Immunostaining, Mouse Assay, Confocal Microscopy

    3) Product Images from "High-density yeast-tiling array reveals previously undiscovered introns and extensive regulation of meiotic splicing"

    Article Title: High-density yeast-tiling array reveals previously undiscovered introns and extensive regulation of meiotic splicing

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0610354104

    Meiosis-specific genes are regulated posttranscriptionally by splicing. PCR products, from primers that surround intronic sequences in 16 meiotic genes (designated above), were separated by using gel electrophoresis on an agarose gel stained with SYBR green. The image color has been inverted for clarity; bands of DNA appear dark on a light background. For each gene, five PCRs were carried out under identical conditions by using five different SK1-derived templates: total RNA (RNA) as a control for genomic contamination, genomic DNA (gDNA), cDNA from cells grown in rich media after 0 hours of sporulation (0h), cDNA from cells harvested after 4 (4h) or 8 (8h) hours of sporulation. The larger PCR products result from genomic DNA in the gDNA lanes or unspliced pre-mRNA in the 0h, 4h, and 8h lanes. The smaller products result from spliced mRNA. The marker (m) is a 50-bp marker. All 13 meiosis-specific genes perform regulated splicing. Only GLC7 , TUB1 , and TUB3 , which have important cellular functions outside of meiosis, are completely spliced in rich media and do not display regulated splicing activity.
    Figure Legend Snippet: Meiosis-specific genes are regulated posttranscriptionally by splicing. PCR products, from primers that surround intronic sequences in 16 meiotic genes (designated above), were separated by using gel electrophoresis on an agarose gel stained with SYBR green. The image color has been inverted for clarity; bands of DNA appear dark on a light background. For each gene, five PCRs were carried out under identical conditions by using five different SK1-derived templates: total RNA (RNA) as a control for genomic contamination, genomic DNA (gDNA), cDNA from cells grown in rich media after 0 hours of sporulation (0h), cDNA from cells harvested after 4 (4h) or 8 (8h) hours of sporulation. The larger PCR products result from genomic DNA in the gDNA lanes or unspliced pre-mRNA in the 0h, 4h, and 8h lanes. The smaller products result from spliced mRNA. The marker (m) is a 50-bp marker. All 13 meiosis-specific genes perform regulated splicing. Only GLC7 , TUB1 , and TUB3 , which have important cellular functions outside of meiosis, are completely spliced in rich media and do not display regulated splicing activity.

    Techniques Used: Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Agarose Gel Electrophoresis, Staining, SYBR Green Assay, Derivative Assay, Marker, Activity Assay

    4) Product Images from "The Smaug RNA-Binding Protein Is Essential for microRNA Synthesis During the Drosophila Maternal-to-Zygotic Transition"

    Article Title: The Smaug RNA-Binding Protein Is Essential for microRNA Synthesis During the Drosophila Maternal-to-Zygotic Transition

    Journal: G3: Genes|Genomes|Genetics

    doi: 10.1534/g3.116.034199

    RT-qPCR shows that Ago1 mRNA expression is unaffected in smg mutants whereas miRNA levels are dramatically reduced. (A) RT-qPCR of Ago1 mRNA shows that expression is unaffected in smg mutant eggs and embryos. (B) RT-qPCR shows that a full-length smg transgene restores expression of miR-1-3p , miR-9-5p , and miR-309-3p expression in 2–4 hr smg 47 mutant embryos. AGO1, Argonaute 1; SMG, Smaug; FL, full-length smg transgene; mRNA, messenger RNA; rep, biological replicate; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; wt, wild type.
    Figure Legend Snippet: RT-qPCR shows that Ago1 mRNA expression is unaffected in smg mutants whereas miRNA levels are dramatically reduced. (A) RT-qPCR of Ago1 mRNA shows that expression is unaffected in smg mutant eggs and embryos. (B) RT-qPCR shows that a full-length smg transgene restores expression of miR-1-3p , miR-9-5p , and miR-309-3p expression in 2–4 hr smg 47 mutant embryos. AGO1, Argonaute 1; SMG, Smaug; FL, full-length smg transgene; mRNA, messenger RNA; rep, biological replicate; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; wt, wild type.

    Techniques Used: Quantitative RT-PCR, Expressing, Mutagenesis, Real-time Polymerase Chain Reaction

    5) Product Images from "CC Chemokine Ligand 3 (CCL3) Regulates CD8+-T-Cell Effector Function and Migration following Viral Infection"

    Article Title: CC Chemokine Ligand 3 (CCL3) Regulates CD8+-T-Cell Effector Function and Migration following Viral Infection

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.7.4004-4014.2003

    Chemokine receptor gene expression in CCL3 +/+ and CCL3 −/− mice. (A) CCR7, CXCR3, and CCR5 mRNA transcript levels were determined by RPA analysis of total RNA obtained from CD8 + T cells isolated from CLN of MHV-infected CCL3 +/+ and CCL3 −/− mice at 7 and 12 days p.i. (B and C) Densitometric analysis of chemokine receptor mRNA transcripts obtained from the scanned autoradiograph. CCL3 −/− CD8 + T cells exhibited a reduction in transcript levels for CXCR3 and CCR5 and an increase in CCR7 expression at both time points. Data are the average normalized units representing the ratio of band intensity to L32 control. Nine CCL3 +/+ mice and nine CCL3 −/− mice were used. A similar receptor profile was observed in spleens of CCL3 +/+ and CCL3 −/− mice (data not shown). Sham-infected CCL3 +/+ and CCL3 −/− mice displayed receptor profiles similar to those of MHV-infected CCL3 −/− mice at day 7 p.i. (data not shown).
    Figure Legend Snippet: Chemokine receptor gene expression in CCL3 +/+ and CCL3 −/− mice. (A) CCR7, CXCR3, and CCR5 mRNA transcript levels were determined by RPA analysis of total RNA obtained from CD8 + T cells isolated from CLN of MHV-infected CCL3 +/+ and CCL3 −/− mice at 7 and 12 days p.i. (B and C) Densitometric analysis of chemokine receptor mRNA transcripts obtained from the scanned autoradiograph. CCL3 −/− CD8 + T cells exhibited a reduction in transcript levels for CXCR3 and CCR5 and an increase in CCR7 expression at both time points. Data are the average normalized units representing the ratio of band intensity to L32 control. Nine CCL3 +/+ mice and nine CCL3 −/− mice were used. A similar receptor profile was observed in spleens of CCL3 +/+ and CCL3 −/− mice (data not shown). Sham-infected CCL3 +/+ and CCL3 −/− mice displayed receptor profiles similar to those of MHV-infected CCL3 −/− mice at day 7 p.i. (data not shown).

    Techniques Used: Expressing, Mouse Assay, Recombinase Polymerase Amplification, Isolation, Infection, Autoradiography

    6) Product Images from "Control of stability of cyclin D1 by quinone reductase 2 in CWR22Rv1 prostate cancer cells"

    Article Title: Control of stability of cyclin D1 by quinone reductase 2 in CWR22Rv1 prostate cancer cells

    Journal: Carcinogenesis

    doi: 10.1093/carcin/bgs016

    Analysis of stability of cyclin D1 protein in relation to NQO2 knockdown. ( A ) Effect of cycloheximide on turnover/degradation of cyclin D1 in shRNA08 or shRNA25. Cells were treated with 30 μg/ml cycloheximide to inhibit new protein synthesis for the times indicated and changes in cyclin D1 were analyzed by western blot analysis, with actin expression used as loading control. Cyclin D1 expression was quantified by densitometric analysis and the expression was presented as percentage (%) remaining relative to time 0. ( B ) Effect of knockdown NQO2 on proteasome activity. Both shRNA08 and shRNA25 cells were treated with or without the addition of proteasome inhibitor MG132 (5 μM) for 4 h. Changes in proteasome activity were measured using chymotrypsin-like proteasome activity assay kit obtained from Promega. ( C ) Effect of proteasome inhibitor MG132 on degradation of phosphorylated cyclin D1 (Thr286) and cyclin D1 in shRNA08 versus shRNA25. Cells were treated with 5 μM MG132 for 0, 2, 4 and 8 h and changes in protein expression were analyzed by western blot analysis, with actin used as loading control. ( D ) Effect of proteasome inhibitor MG132 on degradation of GSk-3β and AKT in shRNA08 versus shRNA25. Cells were treated with 5 μM MG132 for 0, 2, 4 and 8 h and changes in GSk-3β and AKT expression were analyzed by western blot analysis, using actin as loading control. ( E ) AKT kinase activity assay was performed and changes of phosphor-GSK-3α/β (Ser21/9) expression in shRNA08 and shRNA25 cells at 72 h were analyzed by western blot analysis. ( F ) Reverse transcription–PCR was performed to assess cyclin D1 messenger RNA changes in shRNA08 and shRNA25 cells at 72 h. The expression of GAPDH was used as internal control.
    Figure Legend Snippet: Analysis of stability of cyclin D1 protein in relation to NQO2 knockdown. ( A ) Effect of cycloheximide on turnover/degradation of cyclin D1 in shRNA08 or shRNA25. Cells were treated with 30 μg/ml cycloheximide to inhibit new protein synthesis for the times indicated and changes in cyclin D1 were analyzed by western blot analysis, with actin expression used as loading control. Cyclin D1 expression was quantified by densitometric analysis and the expression was presented as percentage (%) remaining relative to time 0. ( B ) Effect of knockdown NQO2 on proteasome activity. Both shRNA08 and shRNA25 cells were treated with or without the addition of proteasome inhibitor MG132 (5 μM) for 4 h. Changes in proteasome activity were measured using chymotrypsin-like proteasome activity assay kit obtained from Promega. ( C ) Effect of proteasome inhibitor MG132 on degradation of phosphorylated cyclin D1 (Thr286) and cyclin D1 in shRNA08 versus shRNA25. Cells were treated with 5 μM MG132 for 0, 2, 4 and 8 h and changes in protein expression were analyzed by western blot analysis, with actin used as loading control. ( D ) Effect of proteasome inhibitor MG132 on degradation of GSk-3β and AKT in shRNA08 versus shRNA25. Cells were treated with 5 μM MG132 for 0, 2, 4 and 8 h and changes in GSk-3β and AKT expression were analyzed by western blot analysis, using actin as loading control. ( E ) AKT kinase activity assay was performed and changes of phosphor-GSK-3α/β (Ser21/9) expression in shRNA08 and shRNA25 cells at 72 h were analyzed by western blot analysis. ( F ) Reverse transcription–PCR was performed to assess cyclin D1 messenger RNA changes in shRNA08 and shRNA25 cells at 72 h. The expression of GAPDH was used as internal control.

    Techniques Used: Western Blot, Expressing, Activity Assay, Kinase Assay, Polymerase Chain Reaction

    7) Product Images from "Dysregulation of Receptor Interacting Protein-2 and Caspase Recruitment Domain Only Protein Mediates Aberrant Caspase-1 Activation in Huntington's Disease"

    Article Title: Dysregulation of Receptor Interacting Protein-2 and Caspase Recruitment Domain Only Protein Mediates Aberrant Caspase-1 Activation in Huntington's Disease

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.4181-05.2005

    Cop/Rip2 knock-down results in increased/reduced vulnerability to cell death. Mutant-htt ST14A cells were transfected with pU6-siRNA, pU6-siRNA Rip2 S1, pU6-si RNA Rip2 S4, pU6-siRNA Cop S1, or pU6-siRNA Cop S2 and cotransfected with GFP by Lipofectamine. The pU6-siRNA Rip2 S4 and pU6-siRNA Cop S2 constructs are effective at knocking down their respective messages and protein, whereas pU6-siRNA Rip2 S1 and pU6-siRNA Cop S1 are not effective and served as controls. A , At 38–40 h (mutant-htt ST14A cells) after transfection, cells were either extracted for RT-PCR or lysed and either directly analyzed by WB with anti-Rip2 (1:1000) antibodies or immunoprecipitated and analyzed by immunoblot with anti-Cop antibodies (1:100). GAPDH is the internal control for the RT-PCR, β-actin (1:5000) for the Rip2 immunoblot, and the IgG band for the Cop immunoprecipitation immunoblot. B , For apoptotic induction, cells were shifted to 37°C for 14 h. Transfected green cells were scored as either alive (flat) or dead (round)(arrows) under fluorescence microscopy, and deconvoluted images were recorded. C , Cell viability was assessed by the MTS assay, and percentage cell death was determined by visual inspection. Results are representative from three independent experiments. Quantification of cell death was data represent the mean±SD. n ≥3; * p
    Figure Legend Snippet: Cop/Rip2 knock-down results in increased/reduced vulnerability to cell death. Mutant-htt ST14A cells were transfected with pU6-siRNA, pU6-siRNA Rip2 S1, pU6-si RNA Rip2 S4, pU6-siRNA Cop S1, or pU6-siRNA Cop S2 and cotransfected with GFP by Lipofectamine. The pU6-siRNA Rip2 S4 and pU6-siRNA Cop S2 constructs are effective at knocking down their respective messages and protein, whereas pU6-siRNA Rip2 S1 and pU6-siRNA Cop S1 are not effective and served as controls. A , At 38–40 h (mutant-htt ST14A cells) after transfection, cells were either extracted for RT-PCR or lysed and either directly analyzed by WB with anti-Rip2 (1:1000) antibodies or immunoprecipitated and analyzed by immunoblot with anti-Cop antibodies (1:100). GAPDH is the internal control for the RT-PCR, β-actin (1:5000) for the Rip2 immunoblot, and the IgG band for the Cop immunoprecipitation immunoblot. B , For apoptotic induction, cells were shifted to 37°C for 14 h. Transfected green cells were scored as either alive (flat) or dead (round)(arrows) under fluorescence microscopy, and deconvoluted images were recorded. C , Cell viability was assessed by the MTS assay, and percentage cell death was determined by visual inspection. Results are representative from three independent experiments. Quantification of cell death was data represent the mean±SD. n ≥3; * p

    Techniques Used: Mutagenesis, Transfection, Construct, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunoprecipitation, Fluorescence, Microscopy, MTS Assay

    8) Product Images from "MicroRNA-146 inhibits pro-inflammatory cytokine secretion through IL-1 receptor-associated kinase 1 in human gingival fibroblasts"

    Article Title: MicroRNA-146 inhibits pro-inflammatory cytokine secretion through IL-1 receptor-associated kinase 1 in human gingival fibroblasts

    Journal: Journal of Inflammation (London, England)

    doi: 10.1186/1476-9255-10-20

    The viability of HGFs after miRNA-146 inhibition. ( A ) HGFs were transfected with 10 and 100 nM miRNA-146a and miRNA-146b-5p and stimulated with 1 μg/ml of P.g LPS for 24 hours. Total RNA from the HGFs was harvested at different times for the quantitative RT-PCR assays. ( B ) The HGFs were collected and separated by FACS based on PI and Annexin V labeling. The numbers indicate the ratios of necrosis and apoptosis. The results shown represent one of three independent experiments.
    Figure Legend Snippet: The viability of HGFs after miRNA-146 inhibition. ( A ) HGFs were transfected with 10 and 100 nM miRNA-146a and miRNA-146b-5p and stimulated with 1 μg/ml of P.g LPS for 24 hours. Total RNA from the HGFs was harvested at different times for the quantitative RT-PCR assays. ( B ) The HGFs were collected and separated by FACS based on PI and Annexin V labeling. The numbers indicate the ratios of necrosis and apoptosis. The results shown represent one of three independent experiments.

    Techniques Used: Inhibition, Transfection, Quantitative RT-PCR, FACS, Labeling

    miRNA-146a and miRNA-146b-5p increase after P.g LPS stimulation in HGFs. ( A ) The fold change of the miRNAs from the miRNA microarray is shown in the schematic diagram. After the HGFs were cultured in the presence or absence of 1 μg/ml of P.g LPS for 24 hours, total RNA was collected and used for the miRNA array. The fold change is calculated by dividing the LPS-stimulated samples by the LPS unstimulated normalized samples. The left red bars show the up-regulated miRNAs, and the right blue bars show the down-regulated miRNAs. The colour difference shows the fold change (up-regulation: from 5.0 to 1.0; down-regulation: from 1.0 to 4.0). ( B ) The expression levels of miRNA-146a and miRNA-146b-5p in the pooled RNA samples using real-time PCR. The relative expression levels are presented as the fold change between the LPS-stimulated (24 hour stimulation with 1 μg/ml of P.g LPS) and unstimulated HGFs. Black bars refer to microarray results, and grey bars refer to the real-time quantitative RT-PCR results. ( C ) The expression levels of miRNA-146a and miRNA-146b-5p in 5 individual RNA samples using real-time PCR. The relative levels are presented as the fold change between the LPS-stimulated and unstimulated HGFs. The short transverse lines indicate the average values. **: p
    Figure Legend Snippet: miRNA-146a and miRNA-146b-5p increase after P.g LPS stimulation in HGFs. ( A ) The fold change of the miRNAs from the miRNA microarray is shown in the schematic diagram. After the HGFs were cultured in the presence or absence of 1 μg/ml of P.g LPS for 24 hours, total RNA was collected and used for the miRNA array. The fold change is calculated by dividing the LPS-stimulated samples by the LPS unstimulated normalized samples. The left red bars show the up-regulated miRNAs, and the right blue bars show the down-regulated miRNAs. The colour difference shows the fold change (up-regulation: from 5.0 to 1.0; down-regulation: from 1.0 to 4.0). ( B ) The expression levels of miRNA-146a and miRNA-146b-5p in the pooled RNA samples using real-time PCR. The relative expression levels are presented as the fold change between the LPS-stimulated (24 hour stimulation with 1 μg/ml of P.g LPS) and unstimulated HGFs. Black bars refer to microarray results, and grey bars refer to the real-time quantitative RT-PCR results. ( C ) The expression levels of miRNA-146a and miRNA-146b-5p in 5 individual RNA samples using real-time PCR. The relative levels are presented as the fold change between the LPS-stimulated and unstimulated HGFs. The short transverse lines indicate the average values. **: p

    Techniques Used: Microarray, Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    9) Product Images from "Surfactant Protein D Reverses the Gene Signature of Transepithelial HIV-1 Passage and Restricts the Viral Transfer Across the Vaginal Barrier"

    Article Title: Surfactant Protein D Reverses the Gene Signature of Transepithelial HIV-1 Passage and Restricts the Viral Transfer Across the Vaginal Barrier

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.00264

    HIV-1 induced global gene signature of EpiVaginal tissues: (A) Gene regulatory network of differentially expressed genes and pathways by the EpiVaginal tissues treated with HIV-1 vs. Untreated EpiVaginal Control tissues. Biological processes are blue colored blocks downregulated genes are green colored, and upregulated are in red. Circles are sized according to their p -value. Genes that were used for validation (CD44, XRCC2, SERPINE1, STX3, CREB1) have been highlighted with a red star in their vicinity. (B) Validation of microarray data by real time qPCR. Ect/E6E7 cells were subjected to identical conditions and treatments as for EpiVaginal tissues. RNA was isolated and cDNA was subjected to real time qPCR. Data represents mean ± S. D of three independent experiments. Fold change in expression of the 5 genes for validation were statistically significant ( p
    Figure Legend Snippet: HIV-1 induced global gene signature of EpiVaginal tissues: (A) Gene regulatory network of differentially expressed genes and pathways by the EpiVaginal tissues treated with HIV-1 vs. Untreated EpiVaginal Control tissues. Biological processes are blue colored blocks downregulated genes are green colored, and upregulated are in red. Circles are sized according to their p -value. Genes that were used for validation (CD44, XRCC2, SERPINE1, STX3, CREB1) have been highlighted with a red star in their vicinity. (B) Validation of microarray data by real time qPCR. Ect/E6E7 cells were subjected to identical conditions and treatments as for EpiVaginal tissues. RNA was isolated and cDNA was subjected to real time qPCR. Data represents mean ± S. D of three independent experiments. Fold change in expression of the 5 genes for validation were statistically significant ( p

    Techniques Used: Microarray, Real-time Polymerase Chain Reaction, Isolation, Expressing

    10) Product Images from "Comparative proteome analysis of Tumor necrosis factor ?-stimulated human Vascular Smooth Muscle Cells in response to melittin"

    Article Title: Comparative proteome analysis of Tumor necrosis factor ?-stimulated human Vascular Smooth Muscle Cells in response to melittin

    Journal: Proteome Science

    doi: 10.1186/1477-5956-11-20

    Validation of the two - dimensional - PAGE data by quantitative RT - PCR and Validation of the protein pathway analysis data. A , expression of selected genes in hVSMCs treated with TNF-α or melittin for 12 h was determined by quantitative RT-PCR. Total RNA was isolated from hVSMCs, reverse transcribed and amplified with the specific primers indicated in under “materials and methods.” β-actin was used as the control. B , Total cell lysates (25 μg) and nuclear lysates of hVSMCs treated for 12 h with either TNF-α or melittin were separated by SDS-PAGE. Proteins were blotted onto a PVDF membrane, probed with specific antibodies, and detected as described under “materials and methods.”
    Figure Legend Snippet: Validation of the two - dimensional - PAGE data by quantitative RT - PCR and Validation of the protein pathway analysis data. A , expression of selected genes in hVSMCs treated with TNF-α or melittin for 12 h was determined by quantitative RT-PCR. Total RNA was isolated from hVSMCs, reverse transcribed and amplified with the specific primers indicated in under “materials and methods.” β-actin was used as the control. B , Total cell lysates (25 μg) and nuclear lysates of hVSMCs treated for 12 h with either TNF-α or melittin were separated by SDS-PAGE. Proteins were blotted onto a PVDF membrane, probed with specific antibodies, and detected as described under “materials and methods.”

    Techniques Used: Polyacrylamide Gel Electrophoresis, Quantitative RT-PCR, Expressing, Isolation, Amplification, SDS Page

    11) Product Images from "Adaptations to chronic rapamycin in mice"

    Article Title: Adaptations to chronic rapamycin in mice

    Journal: Pathobiology of Aging & Age Related Diseases

    doi: 10.3402/pba.v6.31688

    Rapamycin effects on 18S ribosomal RNA in colon of eRapa-fed C57BL/6 mice. (a) Graphic comparison of the normalized intensity values for 18S rRNA as quantified by microarray analysis. (b–d) Graphs showing fold changes in rRNA by qRT-PCR normalized to B2M mRNA in response to 14 and 42 ppm diets in colon (b), small intestine (c), and adipose (d).
    Figure Legend Snippet: Rapamycin effects on 18S ribosomal RNA in colon of eRapa-fed C57BL/6 mice. (a) Graphic comparison of the normalized intensity values for 18S rRNA as quantified by microarray analysis. (b–d) Graphs showing fold changes in rRNA by qRT-PCR normalized to B2M mRNA in response to 14 and 42 ppm diets in colon (b), small intestine (c), and adipose (d).

    Techniques Used: Mouse Assay, Microarray, Quantitative RT-PCR

    12) Product Images from "RSR-2, the Caenorhabditis elegans Ortholog of Human Spliceosomal Component SRm300/SRRM2, Regulates Development by Influencing the Transcriptional Machinery"

    Article Title: RSR-2, the Caenorhabditis elegans Ortholog of Human Spliceosomal Component SRm300/SRRM2, Regulates Development by Influencing the Transcriptional Machinery

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003543

    RNA-Seq of rsr-2(RNAi) and prp-8(RNAi) L3 animals. (A) Venn diagrams of down- and up-regulated transcripts in rsr-2(RNAi) (yellow) and prp-8(RNAi) (blue) L3 animal populations. Overlapping transcripts between the two samples are represented in green. (B) Graph representing the abundance of RNA-Seq reads mapped in intronic regions without internal genes corresponding to control gfp(RNAi) , prp-8(RNAi) , and rsr-2(RNAi) animals. (C) Semiquantitative RT-PCR of wild type and smg-1(r861) worms treated with gfp RNAi control, rsr-2 RNAi and prp-8 RNAi. rpl-12 was used as a positive control for intron retention in NMD-defective animals. act-1 was used as an endogenous control. The black box indicates the accumulation of aberrant mRNAs for dpy-2 and dpy-8 in the smg-1(r861) ; prp-8(RNAi) sample, but not in the smg-1(r861) ; rsr-2(RNAi) sample.
    Figure Legend Snippet: RNA-Seq of rsr-2(RNAi) and prp-8(RNAi) L3 animals. (A) Venn diagrams of down- and up-regulated transcripts in rsr-2(RNAi) (yellow) and prp-8(RNAi) (blue) L3 animal populations. Overlapping transcripts between the two samples are represented in green. (B) Graph representing the abundance of RNA-Seq reads mapped in intronic regions without internal genes corresponding to control gfp(RNAi) , prp-8(RNAi) , and rsr-2(RNAi) animals. (C) Semiquantitative RT-PCR of wild type and smg-1(r861) worms treated with gfp RNAi control, rsr-2 RNAi and prp-8 RNAi. rpl-12 was used as a positive control for intron retention in NMD-defective animals. act-1 was used as an endogenous control. The black box indicates the accumulation of aberrant mRNAs for dpy-2 and dpy-8 in the smg-1(r861) ; prp-8(RNAi) sample, but not in the smg-1(r861) ; rsr-2(RNAi) sample.

    Techniques Used: RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction, Positive Control, Activated Clotting Time Assay

    RSR-2 binds to intronless genes. (A) Chromatin-binding profiles of RNAPII and RSR-2, and RNA-Seq reads in intronless genes. RNA-Seq reads correspond to the N2 mid-L4 stage dataset from the modENCODE consortium [82] . (B) ChIP-qPCR for intronless genes with mouse anti-RNAPII (8WG16), rabbit anti-RSR-2 (Q5092) and unspecific mouse and rabbit IgG antibodies (sc-2025 and sc-2027 respectively). All qPCR values are represented as the percentage of input immunoprecipitated.
    Figure Legend Snippet: RSR-2 binds to intronless genes. (A) Chromatin-binding profiles of RNAPII and RSR-2, and RNA-Seq reads in intronless genes. RNA-Seq reads correspond to the N2 mid-L4 stage dataset from the modENCODE consortium [82] . (B) ChIP-qPCR for intronless genes with mouse anti-RNAPII (8WG16), rabbit anti-RSR-2 (Q5092) and unspecific mouse and rabbit IgG antibodies (sc-2025 and sc-2027 respectively). All qPCR values are represented as the percentage of input immunoprecipitated.

    Techniques Used: Binding Assay, RNA Sequencing Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Immunoprecipitation

    13) Product Images from "LncRNA Expression Profiling of Ischemic Stroke During the Transition From the Acute to Subacute Stage"

    Article Title: LncRNA Expression Profiling of Ischemic Stroke During the Transition From the Acute to Subacute Stage

    Journal: Frontiers in Neurology

    doi: 10.3389/fneur.2019.00036

    RNA-seq analysis of mRNAs and LncRNAs in IS patients. (A) RPKM (reads per kilobase per million) distribution for LncRNAs in all PBMCs from IS patients and healthy controls. (B) Three-dimensional principal component analysis (PCA) of all 15 samples to evaluate the variability of RNA-seq data. The axes represent the principal components (PC1, PC2, and PC3). (C–E) Cluster analysis of differentially expressed LncRNAs and mRNAs of IS patients and healthy controls. (C) Hierarchical clustering analysis indicated 3,009 LncRNAs and 3,982 mRNAs that were differentially expressed between IS patients at 24 h ( n = 5) and healthy controls ( n = 5), and the number decreased to 2,034 and 1,641 at 7 days, respectively (D) . (E) The heatmap shows that 73 LncRNAs and 36 mRNAs were differentially expressed between 24 h and 7 days in IS patients. In the color scheme, red indicates higher expression, and green indicates lower expression.
    Figure Legend Snippet: RNA-seq analysis of mRNAs and LncRNAs in IS patients. (A) RPKM (reads per kilobase per million) distribution for LncRNAs in all PBMCs from IS patients and healthy controls. (B) Three-dimensional principal component analysis (PCA) of all 15 samples to evaluate the variability of RNA-seq data. The axes represent the principal components (PC1, PC2, and PC3). (C–E) Cluster analysis of differentially expressed LncRNAs and mRNAs of IS patients and healthy controls. (C) Hierarchical clustering analysis indicated 3,009 LncRNAs and 3,982 mRNAs that were differentially expressed between IS patients at 24 h ( n = 5) and healthy controls ( n = 5), and the number decreased to 2,034 and 1,641 at 7 days, respectively (D) . (E) The heatmap shows that 73 LncRNAs and 36 mRNAs were differentially expressed between 24 h and 7 days in IS patients. In the color scheme, red indicates higher expression, and green indicates lower expression.

    Techniques Used: RNA Sequencing Assay, Expressing

    14) Product Images from "Engineered Zinc-Finger Proteins Can Compensate Genetic Haploinsufficiency by Transcriptional Activation of the Wild-Type Allele: Application to Willams-Beuren Syndrome and Supravalvular Aortic Stenosis"

    Article Title: Engineered Zinc-Finger Proteins Can Compensate Genetic Haploinsufficiency by Transcriptional Activation of the Wild-Type Allele: Application to Willams-Beuren Syndrome and Supravalvular Aortic Stenosis

    Journal: Human Gene Therapy

    doi: 10.1089/hum.2011.201

    Validation of ELN -ZFP clones. (A) HEK293 cells were transiently transfected with plasmids encoding select ELN -ZFPs and the indicated controls. Total RNA was harvested 48 hours later and q-RT-PCR performed for elastin, VEGF-A, and HIF-1α. q-RT-PCR
    Figure Legend Snippet: Validation of ELN -ZFP clones. (A) HEK293 cells were transiently transfected with plasmids encoding select ELN -ZFPs and the indicated controls. Total RNA was harvested 48 hours later and q-RT-PCR performed for elastin, VEGF-A, and HIF-1α. q-RT-PCR

    Techniques Used: Transfection, Reverse Transcription Polymerase Chain Reaction

    15) Product Images from "The human prostanoid DP receptor stimulates mucin secretion in LS174T cells"

    Article Title: The human prostanoid DP receptor stimulates mucin secretion in LS174T cells

    Journal: British Journal of Pharmacology

    doi: 10.1038/sj.bjp.0703688

    Expression of human prostanoid receptor mRNA in LS174T cells. As described under Methods, RT–PCR was performed using total RNA isolated from cultured LS174T cells, in the presence of reverse transcriptase and employing primers specific for each of the human prostanoid receptors: EP 1 , EP 2 , EP 3 , EP 4 DP, FP, IP and TP (lanes 1, 4, 7, 10, 13, 16, 19 and 22, respectively). Analogous negative control reactions for each primer pair were performed under the same conditions but in the absence of reverse transcriptase (lanes 2, 5, 8, 11, 14, 17, 20 and 23). Positive control PCR reactions were performed for each primer pair using the corresponding receptor cDNA as the template, in order to generate PCR products of the expected size as markers (lanes 3, 6, 9, 12, 15, 18, 21 and 24). PCR products were resolved on a 1.2% agarose gel and visualized by ethidium bromide staining. Results are representative of three independent experiments.
    Figure Legend Snippet: Expression of human prostanoid receptor mRNA in LS174T cells. As described under Methods, RT–PCR was performed using total RNA isolated from cultured LS174T cells, in the presence of reverse transcriptase and employing primers specific for each of the human prostanoid receptors: EP 1 , EP 2 , EP 3 , EP 4 DP, FP, IP and TP (lanes 1, 4, 7, 10, 13, 16, 19 and 22, respectively). Analogous negative control reactions for each primer pair were performed under the same conditions but in the absence of reverse transcriptase (lanes 2, 5, 8, 11, 14, 17, 20 and 23). Positive control PCR reactions were performed for each primer pair using the corresponding receptor cDNA as the template, in order to generate PCR products of the expected size as markers (lanes 3, 6, 9, 12, 15, 18, 21 and 24). PCR products were resolved on a 1.2% agarose gel and visualized by ethidium bromide staining. Results are representative of three independent experiments.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Cell Culture, Negative Control, Positive Control, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining

    16) Product Images from "A T7 autogene-based hybrid mRNA/DNA system for long-term shRNA expression in cytoplasm without inefficient nuclear entry"

    Article Title: A T7 autogene-based hybrid mRNA/DNA system for long-term shRNA expression in cytoplasm without inefficient nuclear entry

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-39407-8

    Prolonged silencing effects on target RFP gene in auto_shRFP@LS-transfected B16F10/RFP cells. ( A ) Sustained decrease of RFP mRNA in auto_shRFP@LS-transfected B16F10/RFP cells. For the indicated period of time, B16F10/RFP cells were transfected with synthetic siRFP@LS (corresponding to 50 nM of siRFP), auto_shRFP@LS (15 μg/mL), or auto(−)_shRFP@LS (14.04 μg/mL; corresponding to T7 autogene plasmid-lacking auto_shRFP system). For each RNA samples, the amounts of RFP mRNA were quantitatively estimated by qRT-PCR method, and then plotted against incubation time after transfection. For comparison, each amount of amplified RFP DNA products was normalized with respect to that of amplified β-actin DNA products. Data represent the mean ± s.d. (n = 5). ( B ) Sustained suppression of RFP expression in auto_shRFP@LS-transfected B16F10/RFP cells. After B16F10/RFP cells were transfected with auto_shRFP@LS or synthetic siRFP@LS for the specified period of time, the amounts of RFP protein within each protein samples were determined by immunoblotting method, and then plotted against incubation time after transfection. Data represent the mean ± s.d. (n = 5). The full blot images are presented in Supplementary Fig. S5 . ( C ) Flow cytometry profiles demonstrating the sustained suppression of RFP expression. For the indicated period of time, B16F10/RFP cells were transfected in the same manner as ( A ). RFP-expressing cells were sorted with respect to a prefixed gate region for RFP fluorescence, and then their percentage was plotted against incubation time after transfection. Data represent the mean ± s.d. (n = 3). ( D , E ) A comparison of duration of silencing effect between auto_shRFP system and nuclear shRNA expression system. B16F10/RFP cells were transfected with auto_shRFP@LS (15 μg/mL) or pSuper_shRFP plasmid@LS (15 μg/mL; corresponding to nuclear H1 promoter-driven shRFP expression system), and then relative amounts of RFP mRNA within the cells were plotted versus transfection time ( D ). The representative FACS profiles on 3 days and 4 days post-transfection are presented ( E ).
    Figure Legend Snippet: Prolonged silencing effects on target RFP gene in auto_shRFP@LS-transfected B16F10/RFP cells. ( A ) Sustained decrease of RFP mRNA in auto_shRFP@LS-transfected B16F10/RFP cells. For the indicated period of time, B16F10/RFP cells were transfected with synthetic siRFP@LS (corresponding to 50 nM of siRFP), auto_shRFP@LS (15 μg/mL), or auto(−)_shRFP@LS (14.04 μg/mL; corresponding to T7 autogene plasmid-lacking auto_shRFP system). For each RNA samples, the amounts of RFP mRNA were quantitatively estimated by qRT-PCR method, and then plotted against incubation time after transfection. For comparison, each amount of amplified RFP DNA products was normalized with respect to that of amplified β-actin DNA products. Data represent the mean ± s.d. (n = 5). ( B ) Sustained suppression of RFP expression in auto_shRFP@LS-transfected B16F10/RFP cells. After B16F10/RFP cells were transfected with auto_shRFP@LS or synthetic siRFP@LS for the specified period of time, the amounts of RFP protein within each protein samples were determined by immunoblotting method, and then plotted against incubation time after transfection. Data represent the mean ± s.d. (n = 5). The full blot images are presented in Supplementary Fig. S5 . ( C ) Flow cytometry profiles demonstrating the sustained suppression of RFP expression. For the indicated period of time, B16F10/RFP cells were transfected in the same manner as ( A ). RFP-expressing cells were sorted with respect to a prefixed gate region for RFP fluorescence, and then their percentage was plotted against incubation time after transfection. Data represent the mean ± s.d. (n = 3). ( D , E ) A comparison of duration of silencing effect between auto_shRFP system and nuclear shRNA expression system. B16F10/RFP cells were transfected with auto_shRFP@LS (15 μg/mL) or pSuper_shRFP plasmid@LS (15 μg/mL; corresponding to nuclear H1 promoter-driven shRFP expression system), and then relative amounts of RFP mRNA within the cells were plotted versus transfection time ( D ). The representative FACS profiles on 3 days and 4 days post-transfection are presented ( E ).

    Techniques Used: Transfection, Plasmid Preparation, Quantitative RT-PCR, Incubation, Amplification, Expressing, Flow Cytometry, Cytometry, Fluorescence, shRNA, FACS

    Sustained production of siRNA by higher and more sustained levels of T7 RNA polymerase. ( A ) Co-delivery of T7pol mRNA and T7 autogene induces higher and more sustained levels of T7pol expression than pCMV-triggering T7 autogene. B16F10/RFP cells were transfected by lipoplexes containing T7 autogene plasmids with either T7pol mRNAs or pCMV_T7pol plasmids for the specified period of time, and then cell lysates were subjected to immunoblot staining, based on the anti-T7 RNA polymerase polyclonal antibodies. Each band intensities were quantitatively analyzed using Image J software and EZ-Capture MG. Data represent the mean ± s.d. (n = 5). The full blot image is presented in Supplementary Fig. S4 . ( B ) TaqMan standard curve of synthetic siRFP oligoes versus CT value. siRFP-specific small RNA TaqMan assay was carried out with serial dilution of synthetic siRFP oligoes. The number of siRFP molecules corresponding to each point was calculated and plotted with respect to CT value. ( C ) Quantitative measurement of siRFP molecules processed from shRFP hairpins. B16F10/RFP cells were transfected with auto_shRFP@LS or auto(−)_shRFP@LS for the indicated period of time, and then siRFP-specific small RNA TaqMan assay was performed against the RNA samples isolated from the cell lysates. Based on the standard curve in ( B ) and CT values obtained from siRFP amplification, the amounts of siRFP molecules produced could be calculated, and then plotted against incubation time after transfection. Data represent the mean ± s.d. (n = 5).
    Figure Legend Snippet: Sustained production of siRNA by higher and more sustained levels of T7 RNA polymerase. ( A ) Co-delivery of T7pol mRNA and T7 autogene induces higher and more sustained levels of T7pol expression than pCMV-triggering T7 autogene. B16F10/RFP cells were transfected by lipoplexes containing T7 autogene plasmids with either T7pol mRNAs or pCMV_T7pol plasmids for the specified period of time, and then cell lysates were subjected to immunoblot staining, based on the anti-T7 RNA polymerase polyclonal antibodies. Each band intensities were quantitatively analyzed using Image J software and EZ-Capture MG. Data represent the mean ± s.d. (n = 5). The full blot image is presented in Supplementary Fig. S4 . ( B ) TaqMan standard curve of synthetic siRFP oligoes versus CT value. siRFP-specific small RNA TaqMan assay was carried out with serial dilution of synthetic siRFP oligoes. The number of siRFP molecules corresponding to each point was calculated and plotted with respect to CT value. ( C ) Quantitative measurement of siRFP molecules processed from shRFP hairpins. B16F10/RFP cells were transfected with auto_shRFP@LS or auto(−)_shRFP@LS for the indicated period of time, and then siRFP-specific small RNA TaqMan assay was performed against the RNA samples isolated from the cell lysates. Based on the standard curve in ( B ) and CT values obtained from siRFP amplification, the amounts of siRFP molecules produced could be calculated, and then plotted against incubation time after transfection. Data represent the mean ± s.d. (n = 5).

    Techniques Used: Expressing, Transfection, Staining, Software, TaqMan Assay, Serial Dilution, Isolation, Amplification, Produced, Incubation

    Schematic diagram of T7 autogene-based hybrid mRNA/DNA system and structure of produced shRNA. ( A ) The mechanism of hybrid mRNA/DNA system in the transfected cells is illustrated. 5′-capped T7pol mRNAs are (1) translated into T7pol proteins via cellular translational machinery, and then initial binding (2) of T7pol proteins to T7 promoter on T7 autogene plasmid transcribes IRES-fused T7pol mRNA (3), which is subsequently (1) translated into T7pol protein via cellular translational machinery. After T7pol proteins trigger the autocatalytic positive feedback loop, their iterative binding to T7 promoters on T7 autogene plasmids results in the large quantities of T7pol proteins. A portion of T7pol proteins exiting the autocatalytic loop bind to T7 promoters of pT7/shRNA DNA fragments, and induce the expression of shRNA in the cytoplasm. ( B ) The predicted hairpin structure of RNA expressed from pT7/shRNA DNA fragment. The RNA sequences of hairpin directed against red fluorescence protein (RFP) mRNA are depicted: 19-nt sequences (blue) from the target mRNA are separated by a short loop (green) from the reverse complement (red) of the same sequences. The mfold web server was used for simulation of RNA folding ( http://mfold.rna.albany.edu/?q=mfold/RNA-Folding-Form ).
    Figure Legend Snippet: Schematic diagram of T7 autogene-based hybrid mRNA/DNA system and structure of produced shRNA. ( A ) The mechanism of hybrid mRNA/DNA system in the transfected cells is illustrated. 5′-capped T7pol mRNAs are (1) translated into T7pol proteins via cellular translational machinery, and then initial binding (2) of T7pol proteins to T7 promoter on T7 autogene plasmid transcribes IRES-fused T7pol mRNA (3), which is subsequently (1) translated into T7pol protein via cellular translational machinery. After T7pol proteins trigger the autocatalytic positive feedback loop, their iterative binding to T7 promoters on T7 autogene plasmids results in the large quantities of T7pol proteins. A portion of T7pol proteins exiting the autocatalytic loop bind to T7 promoters of pT7/shRNA DNA fragments, and induce the expression of shRNA in the cytoplasm. ( B ) The predicted hairpin structure of RNA expressed from pT7/shRNA DNA fragment. The RNA sequences of hairpin directed against red fluorescence protein (RFP) mRNA are depicted: 19-nt sequences (blue) from the target mRNA are separated by a short loop (green) from the reverse complement (red) of the same sequences. The mfold web server was used for simulation of RNA folding ( http://mfold.rna.albany.edu/?q=mfold/RNA-Folding-Form ).

    Techniques Used: Produced, shRNA, Transfection, Binding Assay, Plasmid Preparation, Expressing, Fluorescence

    17) Product Images from "Clock Genes Show Circadian Rhythms in Salivary Glands"

    Article Title: Clock Genes Show Circadian Rhythms in Salivary Glands

    Journal: Journal of Dental Research

    doi: 10.1177/0022034512451450

    Expression of potential downstream targets of clock genes. Analysis of real-time PCR data showed that Aqp5 RNA is expressed in a circadian manner in SGs under light/dark and dark/dark conditions ( A, B ). In contrast, Ae2a showed a circadian pattern only
    Figure Legend Snippet: Expression of potential downstream targets of clock genes. Analysis of real-time PCR data showed that Aqp5 RNA is expressed in a circadian manner in SGs under light/dark and dark/dark conditions ( A, B ). In contrast, Ae2a showed a circadian pattern only

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    18) Product Images from "Trypanosome cdc2-Related Kinase 9 Controls Spliced Leader RNA cap4 Methylation and Phosphorylation of RNA Polymerase II Subunit RPB1"

    Article Title: Trypanosome cdc2-Related Kinase 9 Controls Spliced Leader RNA cap4 Methylation and Phosphorylation of RNA Polymerase II Subunit RPB1

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00156-13

    RPB1 phosphorylation and SL RNA cap4 formation depend on CRK9 kinase activity. (A and B) A CRK9 RNAi parent cell line (A) was generated in which the inducibly expressed dsRNA targets the 3′-UTR of CRK9 mRNA. This cell line was further modified
    Figure Legend Snippet: RPB1 phosphorylation and SL RNA cap4 formation depend on CRK9 kinase activity. (A and B) A CRK9 RNAi parent cell line (A) was generated in which the inducibly expressed dsRNA targets the 3′-UTR of CRK9 mRNA. This cell line was further modified

    Techniques Used: Activity Assay, Generated, Modification

    19) Product Images from "Deoxycytidine kinase promotes the migration and invasion of fibroblast-like synoviocytes from rheumatoid arthritis patients"

    Article Title: Deoxycytidine kinase promotes the migration and invasion of fibroblast-like synoviocytes from rheumatoid arthritis patients

    Journal: International Journal of Clinical and Experimental Pathology

    doi:

    Effect of DCK knockdown on MMPs production in RA FLS. A-C: Total RNA from the DCK-shRNA cells and the control cells were used to analyze the mRNA expression levels of MMP1, MMP3, TIMP-2 by qPCR. D and E: Protein production of MMP-1 and MMP-3 were measured
    Figure Legend Snippet: Effect of DCK knockdown on MMPs production in RA FLS. A-C: Total RNA from the DCK-shRNA cells and the control cells were used to analyze the mRNA expression levels of MMP1, MMP3, TIMP-2 by qPCR. D and E: Protein production of MMP-1 and MMP-3 were measured

    Techniques Used: shRNA, Expressing, Real-time Polymerase Chain Reaction

    20) Product Images from "TGEV infection up-regulates FcRn expression via activation of NF-κB signaling"

    Article Title: TGEV infection up-regulates FcRn expression via activation of NF-κB signaling

    Journal: Scientific Reports

    doi: 10.1038/srep32154

    Effect of NF-κB inhibitors on the expression of pFcRn. IPEC-J2 cells were incubated with or without the NF-κB -specific inhibitor BAY 11-7082 (10 μM) for 30 min. IPEC-J2 cells were subsequently infected with or without TGEV. At the end of the incubation periods for the indicated times, RNA was isolated and analyzed by quantitative real-time RT-PCR.
    Figure Legend Snippet: Effect of NF-κB inhibitors on the expression of pFcRn. IPEC-J2 cells were incubated with or without the NF-κB -specific inhibitor BAY 11-7082 (10 μM) for 30 min. IPEC-J2 cells were subsequently infected with or without TGEV. At the end of the incubation periods for the indicated times, RNA was isolated and analyzed by quantitative real-time RT-PCR.

    Techniques Used: Expressing, Incubation, Infection, Isolation, Quantitative RT-PCR

    21) Product Images from "Functional and Structural Analysis of a Highly-Expressed Yersinia pestis Small RNA following Infection of Cultured Macrophages"

    Article Title: Functional and Structural Analysis of a Highly-Expressed Yersinia pestis Small RNA following Infection of Cultured Macrophages

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0168915

    Validation of candidate sRNA gene expression. (A) sRNA expression was analyzed by northern blot. Total Y . pestis RNA was separated on a 6% denaturing polyacrylamide gel, transferred to a nylon membrane, and probed with biotinylated oligos to target specific sRNAs. The size of the transcripts was determined by using a polyA-tailed RNA ladder. (B) RT-PCR analysis was performed on total RNA isolated from Y . pestis in the intracellular (IC) or extracellular (EC) fraction after infection of THP-1 cells or grown at 37° or 26°C. The relative sRNA levels are presented as fold expression between either intracellular and extracellular fractions or the two temperature conditions. For each sample, the sRNA levels were normalized to 5S rRNA. The average mean and standard deviation from three representative experiments are shown.
    Figure Legend Snippet: Validation of candidate sRNA gene expression. (A) sRNA expression was analyzed by northern blot. Total Y . pestis RNA was separated on a 6% denaturing polyacrylamide gel, transferred to a nylon membrane, and probed with biotinylated oligos to target specific sRNAs. The size of the transcripts was determined by using a polyA-tailed RNA ladder. (B) RT-PCR analysis was performed on total RNA isolated from Y . pestis in the intracellular (IC) or extracellular (EC) fraction after infection of THP-1 cells or grown at 37° or 26°C. The relative sRNA levels are presented as fold expression between either intracellular and extracellular fractions or the two temperature conditions. For each sample, the sRNA levels were normalized to 5S rRNA. The average mean and standard deviation from three representative experiments are shown.

    Techniques Used: Expressing, Northern Blot, Reverse Transcription Polymerase Chain Reaction, Isolation, Infection, Standard Deviation

    22) Product Images from "Addressing RNA Integrity to Determine the Impact of Mitochondrial DNA Mutations on Brain Mitochondrial Function with Age"

    Article Title: Addressing RNA Integrity to Determine the Impact of Mitochondrial DNA Mutations on Brain Mitochondrial Function with Age

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0096940

    Level of RNA Deletions is Age-Independent. Intentional capture of mtRNA deletions by PCR amplification of extended cDNA regions.
    Figure Legend Snippet: Level of RNA Deletions is Age-Independent. Intentional capture of mtRNA deletions by PCR amplification of extended cDNA regions.

    Techniques Used: Polymerase Chain Reaction, Amplification

    Validation of Method. (A) Flow chart illustrating the procedure for detection of mtRNA integrity. (B) Comparison of RNA integrity in CoxI site obtained by using two different commercially available reverse transcriptases. (C) Estimation of errors introduced in the PCR amplication. The 12S rRNA region was amplified with increasing cycle number and 100 ng PCR product was either digested with TaqI or left untreated and subsequently analyzed for mutations. The mutation frequency is plotted as function of PCR cycle number. The figures show mean with SD from three independent experiments.
    Figure Legend Snippet: Validation of Method. (A) Flow chart illustrating the procedure for detection of mtRNA integrity. (B) Comparison of RNA integrity in CoxI site obtained by using two different commercially available reverse transcriptases. (C) Estimation of errors introduced in the PCR amplication. The 12S rRNA region was amplified with increasing cycle number and 100 ng PCR product was either digested with TaqI or left untreated and subsequently analyzed for mutations. The mutation frequency is plotted as function of PCR cycle number. The figures show mean with SD from three independent experiments.

    Techniques Used: Flow Cytometry, Polymerase Chain Reaction, Amplification, Mutagenesis

    RNA Error Frequency is Age-Independent in Controls but Elevated in Mutator Mice. (A) mtRNA errors were measured as described at the same 7 loci as for mtDNA from brains of young (1 months, n = 8) and old (18 months, n = 8). (B) mtRNA errors in heterozygous (mut/+, n = 3) and homozygous (mut/mut, n = 3) mice. Figures show mean with SD. p**
    Figure Legend Snippet: RNA Error Frequency is Age-Independent in Controls but Elevated in Mutator Mice. (A) mtRNA errors were measured as described at the same 7 loci as for mtDNA from brains of young (1 months, n = 8) and old (18 months, n = 8). (B) mtRNA errors in heterozygous (mut/+, n = 3) and homozygous (mut/mut, n = 3) mice. Figures show mean with SD. p**

    Techniques Used: Mouse Assay

    23) Product Images from "Deep sequencing leads to the identification of eukaryotic translation initiation factor 5A as a key element in Rsv1‐mediated lethal systemic hypersensitive response to Soybean mosaic virus infection in soybean"

    Article Title: Deep sequencing leads to the identification of eukaryotic translation initiation factor 5A as a key element in Rsv1‐mediated lethal systemic hypersensitive response to Soybean mosaic virus infection in soybean

    Journal: Molecular Plant Pathology

    doi: 10.1111/mpp.12407

    Expression levels of GmelF5A induced by G7 infection. (A) Phylogenetic analysis of seven homologous soybean GmeIF5A genes. The numbers at the nodes are percentages of bootstrap values (1000 replicates). The scale bar indicates 0.01 substitutions per nucleotide position. (B) Heat map displaying the hierarchical clustering of the expression patterns of seven soybean eIF5A genes induced by Soybean mosaic virus (SMV) inoculation at 14 days post‐inoculation (dpi). Colours indicate the log‐scaled value of the normalized read count per kilobase of exon model per million reads. (C) The RNA‐seq read density of seven soybean GmeIF5A genes induced by SMV inoculation compared with mock‐inoculated plants. The read density value is the normalized read count per million reads. (D) The related expression level of Glyma02g12520 in systemic leaves was validated by reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR) analysis at 0, 7, 14 and 21 dpi after SMV inoculation. The soybean Actin ( GmACT11 ) gene was used as an internal control. Error bars represent mean ± standard deviation (SD) and the data are averages from three biological replicates. Asterisks indicate statistically significant differences compared with the mock control (Student's t ‐test): *** P
    Figure Legend Snippet: Expression levels of GmelF5A induced by G7 infection. (A) Phylogenetic analysis of seven homologous soybean GmeIF5A genes. The numbers at the nodes are percentages of bootstrap values (1000 replicates). The scale bar indicates 0.01 substitutions per nucleotide position. (B) Heat map displaying the hierarchical clustering of the expression patterns of seven soybean eIF5A genes induced by Soybean mosaic virus (SMV) inoculation at 14 days post‐inoculation (dpi). Colours indicate the log‐scaled value of the normalized read count per kilobase of exon model per million reads. (C) The RNA‐seq read density of seven soybean GmeIF5A genes induced by SMV inoculation compared with mock‐inoculated plants. The read density value is the normalized read count per million reads. (D) The related expression level of Glyma02g12520 in systemic leaves was validated by reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR) analysis at 0, 7, 14 and 21 dpi after SMV inoculation. The soybean Actin ( GmACT11 ) gene was used as an internal control. Error bars represent mean ± standard deviation (SD) and the data are averages from three biological replicates. Asterisks indicate statistically significant differences compared with the mock control (Student's t ‐test): *** P

    Techniques Used: Expressing, Infection, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Standard Deviation

    Effects of silencing of GmeIF5A on lethal systemic hypersensitive response (LSHR) severity and Soybean mosaic virus (SMV) G7 viral RNA accumulation in Rsv1 ‐genotype soybean (PI96983). (A) Vector‐only control plants and GmeIF5A virus‐induced gene‐silenced plants were inoculated with SMV G7. LSHR symptoms developed on leaves of both control and GmeIF5A ‐silenced plants at 14 and 21 days post‐inoculation of G7. Vector, inoculated with the empty vector; BPMVR2: eIF5A , inoculated with the eIF5A ‐BPMV vector. (B) Reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR) analyses of the accumulated viral RNA (SMV coat protein, SMV‐CP ) and the expression of pathogenesis‐related 1 ( PR1 ) transcript in the control and GmeIF5A ‐silenced plants at 7, 14 and 21 dpi of G7. The soybean Actin ( GmACT11 ) gene was used as an internal control. Error bars represent mean ± standard deviation (SD) and the data are averages from three independent experiments. Asterisks indicate statistically significant differences between the vector control plant and the GmeIF5A ‐silenced plant at 14 and 21 dpi after G7 inoculation (Student's t ‐test): *** P
    Figure Legend Snippet: Effects of silencing of GmeIF5A on lethal systemic hypersensitive response (LSHR) severity and Soybean mosaic virus (SMV) G7 viral RNA accumulation in Rsv1 ‐genotype soybean (PI96983). (A) Vector‐only control plants and GmeIF5A virus‐induced gene‐silenced plants were inoculated with SMV G7. LSHR symptoms developed on leaves of both control and GmeIF5A ‐silenced plants at 14 and 21 days post‐inoculation of G7. Vector, inoculated with the empty vector; BPMVR2: eIF5A , inoculated with the eIF5A ‐BPMV vector. (B) Reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR) analyses of the accumulated viral RNA (SMV coat protein, SMV‐CP ) and the expression of pathogenesis‐related 1 ( PR1 ) transcript in the control and GmeIF5A ‐silenced plants at 7, 14 and 21 dpi of G7. The soybean Actin ( GmACT11 ) gene was used as an internal control. Error bars represent mean ± standard deviation (SD) and the data are averages from three independent experiments. Asterisks indicate statistically significant differences between the vector control plant and the GmeIF5A ‐silenced plant at 14 and 21 dpi after G7 inoculation (Student's t ‐test): *** P

    Techniques Used: Plasmid Preparation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Standard Deviation

    24) Product Images from "miR-155 induction in microglial cells suppresses Japanese encephalitis virus replication and negatively modulates innate immune responses"

    Article Title: miR-155 induction in microglial cells suppresses Japanese encephalitis virus replication and negatively modulates innate immune responses

    Journal: Journal of Neuroinflammation

    doi: 10.1186/1742-2094-11-97

    Differential expression of NeurimmiRs in human microglial cells after JEV infection. CHME3 cells were mock-infected or infected with JEV. Total RNA was isolated from cells at different time points. (A) Relative abundance of six NeurimmiRs obtained from human miRNA microarray was shown as log 2 fold-change. (B) Levels of miR-155 and miR-146a were determined by qPCR and plotted as relative to that seen in the mock-infected cells. Data were normalized against U6 snRNA. h, hours; JEV, Japanese encephalitis virus; pi, post-infection. *, P
    Figure Legend Snippet: Differential expression of NeurimmiRs in human microglial cells after JEV infection. CHME3 cells were mock-infected or infected with JEV. Total RNA was isolated from cells at different time points. (A) Relative abundance of six NeurimmiRs obtained from human miRNA microarray was shown as log 2 fold-change. (B) Levels of miR-155 and miR-146a were determined by qPCR and plotted as relative to that seen in the mock-infected cells. Data were normalized against U6 snRNA. h, hours; JEV, Japanese encephalitis virus; pi, post-infection. *, P

    Techniques Used: Expressing, Infection, Isolation, Microarray, Real-time Polymerase Chain Reaction

    25) Product Images from "Sendai Virus Targets Inflammatory Responses, as Well as the Interferon-Induced Antiviral State, in a Multifaceted Manner"

    Article Title: Sendai Virus Targets Inflammatory Responses, as Well as the Interferon-Induced Antiviral State, in a Multifaceted Manner

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.14.7903-7913.2003

    Comparison of host mRNA levels in 2fTGH cells infected with various SeVs. (A to C) Parallel cultures of 2fTGH cells were infected with 20 PFU of the various SeVs per cell. Total cytoplasmic RNA was prepared with Trizol at 24 hpi, and equal amounts (10 μg) were used as a template for oligo(dT)-primed [ 33 P]cDNA synthesis. The [ 33 P]cDNA was annealed to triplicate DNAs arrayed on nylon membranes, and the [ 33 P]cDNA bound was quantitated in a PhosphorImager. The graphs show the fold increase in each mRNA relative to the mock control. (D) Schematic representation of the viral mutations and their effects on host gene activation. The C proteins are shown as two telescoping boxes representing the longer (C′ and C) and shorter (Y1 and Y2) C proteins, whose activities during infection, and the mutations investigated, are indicated. The promoter mutation GP42 is thought to exert its effect via mutant leader ( Le ) RNA. The presumed requirement for the various wt genetic elements to prevent host gene activation is shown. The names of the mutant SeVs used are also indicated. Instab., instability; sig., signaling.
    Figure Legend Snippet: Comparison of host mRNA levels in 2fTGH cells infected with various SeVs. (A to C) Parallel cultures of 2fTGH cells were infected with 20 PFU of the various SeVs per cell. Total cytoplasmic RNA was prepared with Trizol at 24 hpi, and equal amounts (10 μg) were used as a template for oligo(dT)-primed [ 33 P]cDNA synthesis. The [ 33 P]cDNA was annealed to triplicate DNAs arrayed on nylon membranes, and the [ 33 P]cDNA bound was quantitated in a PhosphorImager. The graphs show the fold increase in each mRNA relative to the mock control. (D) Schematic representation of the viral mutations and their effects on host gene activation. The C proteins are shown as two telescoping boxes representing the longer (C′ and C) and shorter (Y1 and Y2) C proteins, whose activities during infection, and the mutations investigated, are indicated. The promoter mutation GP42 is thought to exert its effect via mutant leader ( Le ) RNA. The presumed requirement for the various wt genetic elements to prevent host gene activation is shown. The names of the mutant SeVs used are also indicated. Instab., instability; sig., signaling.

    Techniques Used: Infection, Activation Assay, Mutagenesis

    26) Product Images from "Post-transcriptional regulation of gene PA5507 controls PQS concentration in Pseudomonas aeruginosa"

    Article Title: Post-transcriptional regulation of gene PA5507 controls PQS concentration in Pseudomonas aeruginosa

    Journal: Molecular microbiology

    doi: 10.1111/mmi.12963

    Translation of gene PA5507 is dependent on qapR translation. (A) Primer extension analysis of qapR operon transcript. Sequencing reaction mixtures are labeled according to nucleotide (A, T, C, G) and lane ‘Ext’ contains the mixture for the primer extension reaction performed with RNA harvested from a 6 h culture of strain Δ qapR harboring a Δ qapR ’- lacZ translational fusion plasmid. The qapR translational start site is denoted as ‘A ( qapR +1)’. Sequencing reactions were exposed for 18 h and primer extension products were exposed for 1 week. (B) Wild-type strain PAO1 carrying translational fusion plasmids driven by the foreign lacUV5 promoter were grown in LB medium for 6 h. Reporter fusions are indicated below each bar and depicted schematically below the figure. Engineered stop codons are indicated by black octagons. Data are represented as the mean ± SD of results from duplicate assays from three separate experiments.
    Figure Legend Snippet: Translation of gene PA5507 is dependent on qapR translation. (A) Primer extension analysis of qapR operon transcript. Sequencing reaction mixtures are labeled according to nucleotide (A, T, C, G) and lane ‘Ext’ contains the mixture for the primer extension reaction performed with RNA harvested from a 6 h culture of strain Δ qapR harboring a Δ qapR ’- lacZ translational fusion plasmid. The qapR translational start site is denoted as ‘A ( qapR +1)’. Sequencing reactions were exposed for 18 h and primer extension products were exposed for 1 week. (B) Wild-type strain PAO1 carrying translational fusion plasmids driven by the foreign lacUV5 promoter were grown in LB medium for 6 h. Reporter fusions are indicated below each bar and depicted schematically below the figure. Engineered stop codons are indicated by black octagons. Data are represented as the mean ± SD of results from duplicate assays from three separate experiments.

    Techniques Used: Sequencing, Labeling, Plasmid Preparation

    27) Product Images from "Effects of Developmental Deltamethrin Exposure on White Adipose Tissue Gene Expression"

    Article Title: Effects of Developmental Deltamethrin Exposure on White Adipose Tissue Gene Expression

    Journal: Journal of biochemical and molecular toxicology

    doi: 10.1002/jbt.21477

    mRNA gene expression data in WAT of 5-month-old adult male mouse pups from dams exposed to 0, 1, or 3 mg deltamethrin/kg every 3 days during gestation and lactation. Total RNA was isolated from WAT, and mRNA levels were quantified by Quantigene Plex 2.0
    Figure Legend Snippet: mRNA gene expression data in WAT of 5-month-old adult male mouse pups from dams exposed to 0, 1, or 3 mg deltamethrin/kg every 3 days during gestation and lactation. Total RNA was isolated from WAT, and mRNA levels were quantified by Quantigene Plex 2.0

    Techniques Used: Expressing, Isolation

    mRNA gene expression of cytokines in WAT of 5-month-old adult male pups from dams exposed to 0, 1, or 3 mg deltamethrin/kg every 3 days during gestation and lactation. Total RNA was isolated from WAT, and mRNA levels were quantified by Quantigene Plex
    Figure Legend Snippet: mRNA gene expression of cytokines in WAT of 5-month-old adult male pups from dams exposed to 0, 1, or 3 mg deltamethrin/kg every 3 days during gestation and lactation. Total RNA was isolated from WAT, and mRNA levels were quantified by Quantigene Plex

    Techniques Used: Expressing, Isolation

    28) Product Images from "High-throughput sequencing analysis of the expression profile of microRNAs and target genes in mechanical force-induced osteoblastic/cementoblastic differentiation of human periodontal ligament cells"

    Article Title: High-throughput sequencing analysis of the expression profile of microRNAs and target genes in mechanical force-induced osteoblastic/cementoblastic differentiation of human periodontal ligament cells

    Journal: American Journal of Translational Research

    doi:

    Hierarchical clustering analysis, scatter plot, and in vitro validation of differentially expressed miRNAs in stretched and non-stretched periodontal ligament cells (PDLCs). (A) Cluster analysis of 47 differentially expressed miRNAs. Red color represents a relative high expression level. Green color shows a relative low expression level. In the stretched group, 31 miRNAs were significantly upregulated and 16 were downregulated compared with the control group. (B) miRNA scatter plot. Red dots represent significantly upregulated miRNAs in the stretched PDLCs compared with the control cells. Green dots show significantly downregulated miRNAs. Blue dots represent equally-expressed miRNAs. (C) RT-qPCR was applied to verify the expression levels of differentially expressed miRNAs. Consistent with (D) the RNA-Seq results, miR-218-5p, miR-138-5p, miR-221-3p, and miR-132-3p were remarkably upregulated, while miR-133a-3p, miR-145-3p, miR-143-5p, miR-486-3p, and miR-210-3p were significantly downregulated in the stretched group (P
    Figure Legend Snippet: Hierarchical clustering analysis, scatter plot, and in vitro validation of differentially expressed miRNAs in stretched and non-stretched periodontal ligament cells (PDLCs). (A) Cluster analysis of 47 differentially expressed miRNAs. Red color represents a relative high expression level. Green color shows a relative low expression level. In the stretched group, 31 miRNAs were significantly upregulated and 16 were downregulated compared with the control group. (B) miRNA scatter plot. Red dots represent significantly upregulated miRNAs in the stretched PDLCs compared with the control cells. Green dots show significantly downregulated miRNAs. Blue dots represent equally-expressed miRNAs. (C) RT-qPCR was applied to verify the expression levels of differentially expressed miRNAs. Consistent with (D) the RNA-Seq results, miR-218-5p, miR-138-5p, miR-221-3p, and miR-132-3p were remarkably upregulated, while miR-133a-3p, miR-145-3p, miR-143-5p, miR-486-3p, and miR-210-3p were significantly downregulated in the stretched group (P

    Techniques Used: In Vitro, Expressing, Quantitative RT-PCR, RNA Sequencing Assay

    29) Product Images from "Differential control of retrovirus silencing in embryonic cells by proteasomal regulation of the ZFP809 retroviral repressor"

    Article Title: Differential control of retrovirus silencing in embryonic cells by proteasomal regulation of the ZFP809 retroviral repressor

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1620879114

    ZFP809 RNAs are expressed in EC, ES, and differentiated cell lines. ( A ) Exon and intron structure of mRNAs encoding the short form and full-length form of ZFP809. Locations of PCR primers used to amplify cDNA products generic to both forms or specific for the short form or for the full-length form are indicated. ( B ) Analysis of RT-PCR products synthesized using the primer pairs indicated in A , with RNA preparations from the indicated cell lines. DNAs were displayed by agarose gel electrophoresis and stained with ethidium bromide. ( C ) qRT-PCR analysis of total ZFP809 mRNA levels in cell lines of F9 (mouse EC), E14 (mouse ES), and NIH 3T3 (fibroblast) cells, normalized to GAPDH mRNA. Error bars denote standard deviation from the mean of replicate assays ( n = 3).
    Figure Legend Snippet: ZFP809 RNAs are expressed in EC, ES, and differentiated cell lines. ( A ) Exon and intron structure of mRNAs encoding the short form and full-length form of ZFP809. Locations of PCR primers used to amplify cDNA products generic to both forms or specific for the short form or for the full-length form are indicated. ( B ) Analysis of RT-PCR products synthesized using the primer pairs indicated in A , with RNA preparations from the indicated cell lines. DNAs were displayed by agarose gel electrophoresis and stained with ethidium bromide. ( C ) qRT-PCR analysis of total ZFP809 mRNA levels in cell lines of F9 (mouse EC), E14 (mouse ES), and NIH 3T3 (fibroblast) cells, normalized to GAPDH mRNA. Error bars denote standard deviation from the mean of replicate assays ( n = 3).

    Techniques Used: Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Synthesized, Agarose Gel Electrophoresis, Staining, Quantitative RT-PCR, Standard Deviation

    30) Product Images from "p38b and JAK-STAT signaling protect against Invertebrate iridescent virus 6 infection in Drosophila"

    Article Title: p38b and JAK-STAT signaling protect against Invertebrate iridescent virus 6 infection in Drosophila

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007020

    JAK-STAT signaling is required for IIV-6-induced turandot expression and survival from virus infection A) S2* cells were transfected with dsRNA targeting hopscotch , domeless , or Stat92E , and 48 hours later were infected with IIV-6 for 24 hours. RNA was then isolated and TotA induction was quantified by qRT-PCR. Data shown are from three biologically independent assays. Two non-overlapping dsRNAs were used to target each gene. Error bars indicate standard deviation and black bars indicate mean. Statistical analysis was performed comparing control dsRNA ( GFP or mCherry ) transfected cells to target gene knockdowns by two-way ANOVA with corrections for multiple comparisons using the Holm-Sidak method (**** p
    Figure Legend Snippet: JAK-STAT signaling is required for IIV-6-induced turandot expression and survival from virus infection A) S2* cells were transfected with dsRNA targeting hopscotch , domeless , or Stat92E , and 48 hours later were infected with IIV-6 for 24 hours. RNA was then isolated and TotA induction was quantified by qRT-PCR. Data shown are from three biologically independent assays. Two non-overlapping dsRNAs were used to target each gene. Error bars indicate standard deviation and black bars indicate mean. Statistical analysis was performed comparing control dsRNA ( GFP or mCherry ) transfected cells to target gene knockdowns by two-way ANOVA with corrections for multiple comparisons using the Holm-Sidak method (**** p

    Techniques Used: Expressing, Infection, Transfection, Isolation, Quantitative RT-PCR, Standard Deviation

    Turandot genes are expressed upon IIV-6 Infection A) Heatmap of mRNA levels for selected immune-related genes following IIV-6 infection of adult w 1118 flies for 12, 24 and 48 hours assayed in duplicate by NanoString nCounter. RNA was isolated from PBS-injected flies at the same time points. Each data point is a biologically independent sample. A’) Detailed comparison of mRNA levels for Tot A or Tot M from nCounter data. B) Fold induction of all eight Tots from w 1118 flies infected with IIV-6, relative to PBS injected controls, at 6, 12, or 24 hours, quantified by qRT-PCR. n = 3, error bars represent SEM and statistical significance determined by Multiple t-tests with correction for multiple comparisons using the Holm-Sidak method. By this analysis, all Turandots were significantly induced with p values between 0.05 and 0.0003 at all time-points with the exception of TotF (ns) and TotE , which was undetectable (nd). C) S2* cells were infected with IIV-6 and TotA expression was assayed by qRT-PCR at the indicated time points. Significance was determined by two-way ANOVA and Sidak’s multiple comparisons test, comparing the infected sample to its time-matched uninfected control. * p
    Figure Legend Snippet: Turandot genes are expressed upon IIV-6 Infection A) Heatmap of mRNA levels for selected immune-related genes following IIV-6 infection of adult w 1118 flies for 12, 24 and 48 hours assayed in duplicate by NanoString nCounter. RNA was isolated from PBS-injected flies at the same time points. Each data point is a biologically independent sample. A’) Detailed comparison of mRNA levels for Tot A or Tot M from nCounter data. B) Fold induction of all eight Tots from w 1118 flies infected with IIV-6, relative to PBS injected controls, at 6, 12, or 24 hours, quantified by qRT-PCR. n = 3, error bars represent SEM and statistical significance determined by Multiple t-tests with correction for multiple comparisons using the Holm-Sidak method. By this analysis, all Turandots were significantly induced with p values between 0.05 and 0.0003 at all time-points with the exception of TotF (ns) and TotE , which was undetectable (nd). C) S2* cells were infected with IIV-6 and TotA expression was assayed by qRT-PCR at the indicated time points. Significance was determined by two-way ANOVA and Sidak’s multiple comparisons test, comparing the infected sample to its time-matched uninfected control. * p

    Techniques Used: Infection, Isolation, Injection, Quantitative RT-PCR, Expressing

    31) Product Images from "A Novel SR-Related Protein Is Required for the Second Step of Pre-mRNA Splicing"

    Article Title: A Novel SR-Related Protein Is Required for the Second Step of Pre-mRNA Splicing

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.25.8.2969-2980.2005

    SRrp53 can regulate 5′ splice site selection in vivo. In vivo splicing analyses were performed with HeLa cells transiently cotransfected with an adenovirus E1A reporter plasmid and expression plasmids encoding for either mouse SRrp53, hnRNP A1 or SF2/ASF. (A) Diagram of the E1A reporter gene is shown. The alternative 5′ splice sites and splicing events that generates 13S, 12S, and 9S mRNAs are shown schematically. The location of the exon primers used for RT-PCR analysis is shown. (B) HeLa cells were transiently cotransfected with the adenovirus E1A reporter plasmid and the expression constructs for each of the proteins indicated above the gel or the parental plasmid (Control). RNA was harvested at 24 h posttransfection and analyzed by RT-PCR with a labeled forward primer, denaturing PAGE, and autoradiography as described in Materials and Methods. The positions of the unspliced pre-mRNA and of 13S, 12S, and 9S spliced mRNAs are indicated to the right. The 10S and 11S isoforms (*) did not arise from competition between alternative 5′ splice sites. (C) Quantitation of E1A mRNA isoforms in transfected cells is shown. The relative amounts of 13S, 12S, and 9S E1A mRNAs were calculated from the data given in panel B by using a phosphorimager, and the percentage of each isoform is shown. Each experiment was repeated four times, and the data represent averages, with bars indicating standard errors.
    Figure Legend Snippet: SRrp53 can regulate 5′ splice site selection in vivo. In vivo splicing analyses were performed with HeLa cells transiently cotransfected with an adenovirus E1A reporter plasmid and expression plasmids encoding for either mouse SRrp53, hnRNP A1 or SF2/ASF. (A) Diagram of the E1A reporter gene is shown. The alternative 5′ splice sites and splicing events that generates 13S, 12S, and 9S mRNAs are shown schematically. The location of the exon primers used for RT-PCR analysis is shown. (B) HeLa cells were transiently cotransfected with the adenovirus E1A reporter plasmid and the expression constructs for each of the proteins indicated above the gel or the parental plasmid (Control). RNA was harvested at 24 h posttransfection and analyzed by RT-PCR with a labeled forward primer, denaturing PAGE, and autoradiography as described in Materials and Methods. The positions of the unspliced pre-mRNA and of 13S, 12S, and 9S spliced mRNAs are indicated to the right. The 10S and 11S isoforms (*) did not arise from competition between alternative 5′ splice sites. (C) Quantitation of E1A mRNA isoforms in transfected cells is shown. The relative amounts of 13S, 12S, and 9S E1A mRNAs were calculated from the data given in panel B by using a phosphorimager, and the percentage of each isoform is shown. Each experiment was repeated four times, and the data represent averages, with bars indicating standard errors.

    Techniques Used: Selection, In Vivo, Plasmid Preparation, Expressing, Reverse Transcription Polymerase Chain Reaction, Construct, Labeling, Polyacrylamide Gel Electrophoresis, Autoradiography, Quantitation Assay, Transfection

    32) Product Images from "ClpP of Streptococcus mutans Differentially Regulates Expression of Genomic Islands, Mutacin Production, and Antibiotic Tolerance ▿ Differentially Regulates Expression of Genomic Islands, Mutacin Production, and Antibiotic Tolerance ▿ †"

    Article Title: ClpP of Streptococcus mutans Differentially Regulates Expression of Genomic Islands, Mutacin Production, and Antibiotic Tolerance ▿ Differentially Regulates Expression of Genomic Islands, Mutacin Production, and Antibiotic Tolerance ▿ †

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01350-09

    clpP deficiency leads to increased sensitivity to trimethoprim. (A) Expression of dihydrofolate reductase transcript in UA159(pIB184), IBS512(pIB184), and IBS512(pIBC7). RNA was extracted from all three cultures and subjected to cDNA synthesis. Semiquantitative RT-PCR was then used to measure the levels of dihydrofolate reductase transcript in all three cultures. The expression of gyrA was included as an internal control to ensure that equal amounts of RNA were analyzed for each culture. (B) Trimethoprim-containing E strips were aseptically placed on UA159 and IBS512 lawns. The plates were incubated under microaerophilic conditions at 37°C overnight. The MICs for the two cultures were noted.
    Figure Legend Snippet: clpP deficiency leads to increased sensitivity to trimethoprim. (A) Expression of dihydrofolate reductase transcript in UA159(pIB184), IBS512(pIB184), and IBS512(pIBC7). RNA was extracted from all three cultures and subjected to cDNA synthesis. Semiquantitative RT-PCR was then used to measure the levels of dihydrofolate reductase transcript in all three cultures. The expression of gyrA was included as an internal control to ensure that equal amounts of RNA were analyzed for each culture. (B) Trimethoprim-containing E strips were aseptically placed on UA159 and IBS512 lawns. The plates were incubated under microaerophilic conditions at 37°C overnight. The MICs for the two cultures were noted.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Incubation

    33) Product Images from "Transdifferentiation of periodontal ligament-derived stem cells into retinal ganglion-like cells and its microRNA signature"

    Article Title: Transdifferentiation of periodontal ligament-derived stem cells into retinal ganglion-like cells and its microRNA signature

    Journal: Scientific Reports

    doi: 10.1038/srep16429

    microRNA expression analysis of human PDLSC along the retinal induction treatment. Total RNA was collected at Day 0, 10, 17 and 24. Five significant miRNAs from the microarray profile ( hsa-miR-132 , hsa-miR-29b , hsa-miR-30d , hsa-miR-630 and hsa-miR-7 ) were validated using TaqMan PCR approach. snRNA U6 was used for normalization. The relative fold changes were compared to the group at Day 0. The data represented, mean ± standard deviation. * p
    Figure Legend Snippet: microRNA expression analysis of human PDLSC along the retinal induction treatment. Total RNA was collected at Day 0, 10, 17 and 24. Five significant miRNAs from the microarray profile ( hsa-miR-132 , hsa-miR-29b , hsa-miR-30d , hsa-miR-630 and hsa-miR-7 ) were validated using TaqMan PCR approach. snRNA U6 was used for normalization. The relative fold changes were compared to the group at Day 0. The data represented, mean ± standard deviation. * p

    Techniques Used: Expressing, Microarray, Polymerase Chain Reaction, Standard Deviation

    34) Product Images from "microRNA-1 regulates sarcomere formation and suppresses smooth muscle gene expression in the mammalian heart"

    Article Title: microRNA-1 regulates sarcomere formation and suppresses smooth muscle gene expression in the mammalian heart

    Journal: eLife

    doi: 10.7554/eLife.01323

    qPCR to detect the miR-1-2/133a-1 or miR-1-1/133a-2 promoter sequences, or an intergenic genomic sequence, following chromatin immunoprecipitation (ChIP) of RNA polymerase II (RNA Pol II) in P2 wild-type or miR-1 null hearts. DOI: http://dx.doi.org/10.7554/eLife.01323.021
    Figure Legend Snippet: qPCR to detect the miR-1-2/133a-1 or miR-1-1/133a-2 promoter sequences, or an intergenic genomic sequence, following chromatin immunoprecipitation (ChIP) of RNA polymerase II (RNA Pol II) in P2 wild-type or miR-1 null hearts. DOI: http://dx.doi.org/10.7554/eLife.01323.021

    Techniques Used: Real-time Polymerase Chain Reaction, Sequencing, Chromatin Immunoprecipitation

    35) Product Images from "Role of Nox4 and Nox2 in Hyperoxia-Induced Reactive Oxygen Species Generation and Migration of Human Lung Endothelial Cells"

    Article Title: Role of Nox4 and Nox2 in Hyperoxia-Induced Reactive Oxygen Species Generation and Migration of Human Lung Endothelial Cells

    Journal: Antioxidants & Redox Signaling

    doi: 10.1089/ars.2008.2203

    Effect of hyperoxia on mRNA expression of Nox and phox family NADPH oxidases in HPAECs. HPAECs grown to ∼90% confluence were exposed to normoxia (room air) or hyperoxia (95% O 2 ) for 12, 24, 48, and 72 h. Total RNA was extracted and expressions of Nox and phox homologues were quantified by real-time RT-PCR and normalized with 18S rRNA. The values are mean ± S.E.M for three independent experiments. *significantly different from normoxia ( p
    Figure Legend Snippet: Effect of hyperoxia on mRNA expression of Nox and phox family NADPH oxidases in HPAECs. HPAECs grown to ∼90% confluence were exposed to normoxia (room air) or hyperoxia (95% O 2 ) for 12, 24, 48, and 72 h. Total RNA was extracted and expressions of Nox and phox homologues were quantified by real-time RT-PCR and normalized with 18S rRNA. The values are mean ± S.E.M for three independent experiments. *significantly different from normoxia ( p

    Techniques Used: Expressing, Quantitative RT-PCR

    Nox4 siRNA attenuates hyperoxia induced expression of Nox4 in nucleus. HPAECs grown on 35-mm dishes ( A and C ) or glass coverslips ( B ) to ∼50% confluence were transfected with scrambled siRNA (Sc) or Nox4 siRNA (50 n M ) for 48 h. ( A ) Total RNA was extracted and Nox4 mRNA expression was quantified and normalized to 18S rRNA by real-time RT-PCR. Values are average of three independent determinations. ( B ) Sc or Nox4 siRNA transfected cells were exposed to normoxia or hyperoxia (3 h), washed, fixed, permeabilized, and probed with anti-Nox4 antibody (from Dr. Lambeth) or DAPI for nuclear staining and examined by immunofluorescence microscopy using a 60X oil objective. The Nox4 ( red ) and DAPI ( blue ) images show matched cell fields for each condition. A representative image from several independent experiments is shown. ( C ).
    Figure Legend Snippet: Nox4 siRNA attenuates hyperoxia induced expression of Nox4 in nucleus. HPAECs grown on 35-mm dishes ( A and C ) or glass coverslips ( B ) to ∼50% confluence were transfected with scrambled siRNA (Sc) or Nox4 siRNA (50 n M ) for 48 h. ( A ) Total RNA was extracted and Nox4 mRNA expression was quantified and normalized to 18S rRNA by real-time RT-PCR. Values are average of three independent determinations. ( B ) Sc or Nox4 siRNA transfected cells were exposed to normoxia or hyperoxia (3 h), washed, fixed, permeabilized, and probed with anti-Nox4 antibody (from Dr. Lambeth) or DAPI for nuclear staining and examined by immunofluorescence microscopy using a 60X oil objective. The Nox4 ( red ) and DAPI ( blue ) images show matched cell fields for each condition. A representative image from several independent experiments is shown. ( C ).

    Techniques Used: Expressing, Transfection, Quantitative RT-PCR, Staining, Immunofluorescence, Microscopy

    Silencing of Nox2 and Nox4 by siRNA upregulates expression of Nox4 and Nox2, respectively. HPAECs grown on 35-mm dishes to ∼50% confluence were transfected with 50 n M scrambled siRNA or Nox2 siRNA or Nox4 siRNA or p22 phox siRNA for 48 h. ( A and B ) Total RNA was isolated and mRNA levels of Nox2 and Nox4 were quantified by real-time RT-PCR and normalized to 18S rRNA. In parallel experiments, total cell lysates (20 μg protein) from scrambled siRNA or Nox2 siRNA or Nox4 siRNA transfected cells were subjected to SDS-PAGE and Western blotted anti-Nox4 (Santa Cruz, CA) ( A ) or anti-Nox2 ( B ) antibodies. Values are average of three independent experiments. Shown are representative of blots from three separate experiments. ( C ) Total RNA was isolated and mRNA levels of p22 phox were determined by real-time RT-PCR and values are average of three independent experiments.
    Figure Legend Snippet: Silencing of Nox2 and Nox4 by siRNA upregulates expression of Nox4 and Nox2, respectively. HPAECs grown on 35-mm dishes to ∼50% confluence were transfected with 50 n M scrambled siRNA or Nox2 siRNA or Nox4 siRNA or p22 phox siRNA for 48 h. ( A and B ) Total RNA was isolated and mRNA levels of Nox2 and Nox4 were quantified by real-time RT-PCR and normalized to 18S rRNA. In parallel experiments, total cell lysates (20 μg protein) from scrambled siRNA or Nox2 siRNA or Nox4 siRNA transfected cells were subjected to SDS-PAGE and Western blotted anti-Nox4 (Santa Cruz, CA) ( A ) or anti-Nox2 ( B ) antibodies. Values are average of three independent experiments. Shown are representative of blots from three separate experiments. ( C ) Total RNA was isolated and mRNA levels of p22 phox were determined by real-time RT-PCR and values are average of three independent experiments.

    Techniques Used: Expressing, Transfection, Isolation, Quantitative RT-PCR, SDS Page, Western Blot

    36) Product Images from "The First Alcohol Drink Triggers mTORC1-Dependent Synaptic Plasticity in Nucleus Accumbens Dopamine D1 Receptor Neurons"

    Article Title: The First Alcohol Drink Triggers mTORC1-Dependent Synaptic Plasticity in Nucleus Accumbens Dopamine D1 Receptor Neurons

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.2254-15.2016

    D1R activation promotes the translation of GLUA1 , HOMER2 , and PSD-95 . RT-PCR of polysomal RNA ( B ) and total RNA ( C ) in the NAc 30 min after intraperitoneal SKF (5 mg/kg) or vehicle (3% DMSO). A , Isolation of polysomes by sucrose gradient centrifugation.
    Figure Legend Snippet: D1R activation promotes the translation of GLUA1 , HOMER2 , and PSD-95 . RT-PCR of polysomal RNA ( B ) and total RNA ( C ) in the NAc 30 min after intraperitoneal SKF (5 mg/kg) or vehicle (3% DMSO). A , Isolation of polysomes by sucrose gradient centrifugation.

    Techniques Used: Activation Assay, Reverse Transcription Polymerase Chain Reaction, Isolation, Gradient Centrifugation

    37) Product Images from "Identification of new high affinity targets for Roquin based on structural conservation"

    Article Title: Identification of new high affinity targets for Roquin based on structural conservation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky908

    The UCP3 wt element folds into two hairpins and reduces mRNA half-life by interaction with Roquin. ( A ) Predicted lowest free energy secondary structure of the UCP3 wt element by RNAstructure. Nucleotides detected by in-line probing [shown in ( B )] are circled. ( B ) In-line probing analysis of UCP3 wt RNA. The RNA was loaded directly (NR, no reaction), subjected to cleavage by RNase T1 or alkaline hydrolysis ( ¯ OH), or incubated for 40 h at room temperature and pH 8.3 (in-line) prior to Urea PAGE. Paired regions are indicated by identically colored lines. ( C ) Luciferase activity of UCP3 mutants for the identification of motifs essential for gene regulation. Adenine was mutated to cytosine, guanine to uracil and vice versa . Numbers indicate mutated nucleotide positions. Firefly luciferase activity was normalized to Renilla luciferase as internal transfection control. Values are normalized to an empty vector control, without UCP3 3′UTR sequences. Luciferase activity of the UCP3 wt element is indicated as dashed line. n = 3. ( D ) GFP fluorescence of UCP3 wt and double mutant (MUTI/II). GFP-UCP3-fusion constructs were stably integrated into the genome of HeLa cells. GFP fluorescence was measured by flow cytometry. ( E ) Half-life of GFP mRNAs containing the UCP3 wt element (wt) or double mutant (MUTI/II). HeLa cells stably expressing one of the two constructs were treated with 5 μg/μl actinomycin D (ActD). Thereafter, total RNA was isolated at 2 h intervals and GFP mRNA levels quantified by RT-qPCR. GFP values are normalized to the housekeeping gene RPLP0. n = 3. ( F ) Overview of UCP3 constructs used for RNA affinity purification. ( G ) Analysis of Roquin binding to the UCP3 constructs shown in ( F ). For RNA affinity purification HEK293 whole cell lysates were incubated with the different UCP3 RNAs. Roquin-1 and Roquin-2 were visualized by western blot using anti-Roquin antibody. n = 2. ( H ) Western blot of Roquin-1 and Roquin-2 after siRNA-mediated knockdown. Anti-Roquin was used to verify the respective knockdown. Total lane protein is shown as loading control. n = 3. ( I ) Luciferase activity of the UCP3 wt element after siRNA-mediated knockdown of Roquin proteins. Firefly luciferase activity was normalized to Renilla luciferase as internal transfection control. Values are normalized to an empty vector control, without UCP3 3′UTR sequences. n = 3. (**) P -value
    Figure Legend Snippet: The UCP3 wt element folds into two hairpins and reduces mRNA half-life by interaction with Roquin. ( A ) Predicted lowest free energy secondary structure of the UCP3 wt element by RNAstructure. Nucleotides detected by in-line probing [shown in ( B )] are circled. ( B ) In-line probing analysis of UCP3 wt RNA. The RNA was loaded directly (NR, no reaction), subjected to cleavage by RNase T1 or alkaline hydrolysis ( ¯ OH), or incubated for 40 h at room temperature and pH 8.3 (in-line) prior to Urea PAGE. Paired regions are indicated by identically colored lines. ( C ) Luciferase activity of UCP3 mutants for the identification of motifs essential for gene regulation. Adenine was mutated to cytosine, guanine to uracil and vice versa . Numbers indicate mutated nucleotide positions. Firefly luciferase activity was normalized to Renilla luciferase as internal transfection control. Values are normalized to an empty vector control, without UCP3 3′UTR sequences. Luciferase activity of the UCP3 wt element is indicated as dashed line. n = 3. ( D ) GFP fluorescence of UCP3 wt and double mutant (MUTI/II). GFP-UCP3-fusion constructs were stably integrated into the genome of HeLa cells. GFP fluorescence was measured by flow cytometry. ( E ) Half-life of GFP mRNAs containing the UCP3 wt element (wt) or double mutant (MUTI/II). HeLa cells stably expressing one of the two constructs were treated with 5 μg/μl actinomycin D (ActD). Thereafter, total RNA was isolated at 2 h intervals and GFP mRNA levels quantified by RT-qPCR. GFP values are normalized to the housekeeping gene RPLP0. n = 3. ( F ) Overview of UCP3 constructs used for RNA affinity purification. ( G ) Analysis of Roquin binding to the UCP3 constructs shown in ( F ). For RNA affinity purification HEK293 whole cell lysates were incubated with the different UCP3 RNAs. Roquin-1 and Roquin-2 were visualized by western blot using anti-Roquin antibody. n = 2. ( H ) Western blot of Roquin-1 and Roquin-2 after siRNA-mediated knockdown. Anti-Roquin was used to verify the respective knockdown. Total lane protein is shown as loading control. n = 3. ( I ) Luciferase activity of the UCP3 wt element after siRNA-mediated knockdown of Roquin proteins. Firefly luciferase activity was normalized to Renilla luciferase as internal transfection control. Values are normalized to an empty vector control, without UCP3 3′UTR sequences. n = 3. (**) P -value

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Fluorescence, Mutagenesis, Construct, Stable Transfection, Flow Cytometry, Cytometry, Expressing, Isolation, Quantitative RT-PCR, Affinity Purification, Binding Assay, Western Blot

    38) Product Images from "Random Amplification and Pyrosequencing for Identification of Novel Viral Genome Sequences"

    Article Title: Random Amplification and Pyrosequencing for Identification of Novel Viral Genome Sequences

    Journal: Journal of Biomolecular Techniques : JBT

    doi: 10.7171/jbt.12-2301-001

    Random RT-PCR amplification of total RNA extract and PCR amplification of RL library for quality assessment. ( A ) Total RNA extract from IQE7620 viral culture supernatant was amplified using an anchored, random amplification method (Lane 1) or NuGEN Technologies'
    Figure Legend Snippet: Random RT-PCR amplification of total RNA extract and PCR amplification of RL library for quality assessment. ( A ) Total RNA extract from IQE7620 viral culture supernatant was amplified using an anchored, random amplification method (Lane 1) or NuGEN Technologies'

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction

    39) Product Images from "Recurrent internal tandem duplications of BCOR in clear cell sarcoma of the kidney"

    Article Title: Recurrent internal tandem duplications of BCOR in clear cell sarcoma of the kidney

    Journal: Nature Communications

    doi: 10.1038/ncomms9891

    BCOR expression in CCSKs. ( a ) Targeted RT–PCR of a segment of the BCOR transcript (exons 14 and 15) in CCSKs from female patients demonstrated expression of both the wild-type product (491 bp) and a larger product corresponding to the mutant (ITD) allele, confirming expression of the ITD from the active X chromosome. ( b ) Box-and-whisker plot of estimated BCOR transcript abundance from RNA-seq data in ITD-positive CCSKs (ITD+) demonstrating high expression of BCOR in comparison to an ITD-negative CCSK (ITD−), Wilms tumours (Wilms) and assorted soft-tissue sarcomas (STS). UDS with BCOR – CCNB3 fusions also had upregulated BCOR expression. Bar representing 25th–75th percentile, line representing the maximum and minimum values. ( c ) Immunoblot using an antibody to full-length BCOR protein 10 demonstrated a 192-kDa product corresponding to the predicted size of BCOR 10 in ITD-positive CCSK tumours (T) but not in matched normal kidney samples (N). ACTB, beta-actin. ( d ) Haematoxylin-and-eosin-stained section of CCSK showing classic histologic pattern. Immunohistochemistry with BCOR antibody demonstrated strong nuclear staining in the tumour cells in ( e ) CCSKs but not in ( f ) Wilms tumours.
    Figure Legend Snippet: BCOR expression in CCSKs. ( a ) Targeted RT–PCR of a segment of the BCOR transcript (exons 14 and 15) in CCSKs from female patients demonstrated expression of both the wild-type product (491 bp) and a larger product corresponding to the mutant (ITD) allele, confirming expression of the ITD from the active X chromosome. ( b ) Box-and-whisker plot of estimated BCOR transcript abundance from RNA-seq data in ITD-positive CCSKs (ITD+) demonstrating high expression of BCOR in comparison to an ITD-negative CCSK (ITD−), Wilms tumours (Wilms) and assorted soft-tissue sarcomas (STS). UDS with BCOR – CCNB3 fusions also had upregulated BCOR expression. Bar representing 25th–75th percentile, line representing the maximum and minimum values. ( c ) Immunoblot using an antibody to full-length BCOR protein 10 demonstrated a 192-kDa product corresponding to the predicted size of BCOR 10 in ITD-positive CCSK tumours (T) but not in matched normal kidney samples (N). ACTB, beta-actin. ( d ) Haematoxylin-and-eosin-stained section of CCSK showing classic histologic pattern. Immunohistochemistry with BCOR antibody demonstrated strong nuclear staining in the tumour cells in ( e ) CCSKs but not in ( f ) Wilms tumours.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Whisker Assay, RNA Sequencing Assay, Staining, Immunohistochemistry

    40) Product Images from "KSHV induces immunoglobulin rearrangements in mature B lymphocytes"

    Article Title: KSHV induces immunoglobulin rearrangements in mature B lymphocytes

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006967

    A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total RNA was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by RT-PCR. Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p
    Figure Legend Snippet: A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total RNA was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by RT-PCR. Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p

    Techniques Used: Infection, Expressing, Reverse Transcription Polymerase Chain Reaction

    41) Product Images from "Genome-Wide Mutagenesis of Dengue Virus Reveals Plasticity of the NS1 Protein and Enables Generation of Infectious Tagged Reporter Viruses"

    Article Title: Genome-Wide Mutagenesis of Dengue Virus Reveals Plasticity of the NS1 Protein and Enables Generation of Infectious Tagged Reporter Viruses

    Journal: Journal of Virology

    doi: 10.1128/JVI.01455-17

    Characterization of epitope- and reporter-tagged DENV-2 constructs. (A) Guided by the insertional map, a panel of tagged viruses were generated that featured the indicated epitope or reporter protein insertions in capsid, adjacent to the membrane anchor (CAPmem), or NS1, immediately downstream of Lys-174. (B) Huh7.5 cells were electroporated with in vitro -transcribed RNA for the indicated DENV-2 constructs and cultured for 4 days prior to Western blot analysis of NS1 protein. Detection of β-actin served as a loading control. Similarly, supernatants from these cells were cleared by centrifugation and subjected to SDS-PAGE under nonreducing, nondenaturing conditions and Western blotting with anti-NS1 antibody. The numbers below the NS1 Western blots indicate the levels of NS1 protein in whole-cell lysates (normalized to β-actin and expressed as a percentage of wild-type levels [%-WT]; upper panel) and extracellular NS1 protein (expressed as a ratio to intracellular NS1 bands, with the wild-type ratio set to 1 [Ext:Int]; lower panel). (C) Automated immunofluorescence analysis of the proportion of capsid protein-positive Huh-7.5 cells at 4 days postelectroporation with the indicated DENV-2 RNA transcripts. Data are means + the standard deviations (SD; n = 3 for > 1,500 cells/electroporation). (D) Infectivity titers were determined by focus forming unit (FFU) assays at 24 to 120 h postelectroporation of Huh-7.5 cells with the indicated DENV-2 RNA transcripts. Data are means + the SD ( n = 3). The dashed line indicates the limit of detection of the assay.
    Figure Legend Snippet: Characterization of epitope- and reporter-tagged DENV-2 constructs. (A) Guided by the insertional map, a panel of tagged viruses were generated that featured the indicated epitope or reporter protein insertions in capsid, adjacent to the membrane anchor (CAPmem), or NS1, immediately downstream of Lys-174. (B) Huh7.5 cells were electroporated with in vitro -transcribed RNA for the indicated DENV-2 constructs and cultured for 4 days prior to Western blot analysis of NS1 protein. Detection of β-actin served as a loading control. Similarly, supernatants from these cells were cleared by centrifugation and subjected to SDS-PAGE under nonreducing, nondenaturing conditions and Western blotting with anti-NS1 antibody. The numbers below the NS1 Western blots indicate the levels of NS1 protein in whole-cell lysates (normalized to β-actin and expressed as a percentage of wild-type levels [%-WT]; upper panel) and extracellular NS1 protein (expressed as a ratio to intracellular NS1 bands, with the wild-type ratio set to 1 [Ext:Int]; lower panel). (C) Automated immunofluorescence analysis of the proportion of capsid protein-positive Huh-7.5 cells at 4 days postelectroporation with the indicated DENV-2 RNA transcripts. Data are means + the standard deviations (SD; n = 3 for > 1,500 cells/electroporation). (D) Infectivity titers were determined by focus forming unit (FFU) assays at 24 to 120 h postelectroporation of Huh-7.5 cells with the indicated DENV-2 RNA transcripts. Data are means + the SD ( n = 3). The dashed line indicates the limit of detection of the assay.

    Techniques Used: Construct, Generated, In Vitro, Cell Culture, Western Blot, Centrifugation, SDS Page, Immunofluorescence, Electroporation, Infection

    42) Product Images from "SRPX2 regulates colon cancer cell metabolism by miR-192/215 via PI3K-Akt"

    Article Title: SRPX2 regulates colon cancer cell metabolism by miR-192/215 via PI3K-Akt

    Journal: American Journal of Translational Research

    doi:

    MiR-192/miR-215 regulates SRPX2 expression in colon cancer cells. (A) miR-192 and miR-215 are the predicted miRNAs targeting SPRX2. (B) miR-192 and miR-215 expression increased in colon cancer cells. SW116 and HCT-8 cells were transfected miR-192 and miR-215 for 48 h, and then total RNA was extracted for real time RT-PCR. (C) Relative luciferase activity of the indicated SRPX2 reporter construct in SW116 cells. SW116 cells were co-transfected with SRPX2 promoter plasmid or miR-192 and miR-215 for 24 h. The cells were collected according to the protocol and analyzed. (D) miR-192 and miR-215 expression increased in colon cancer cells. SW116 and HCT8 cells were transfected miR-192 and miR-215 for 48 h, and then total RNA was extracted for SPRX2 mRNA analysis by real time RT-PCR. (E) Western blot assays were performed to detect the expression of SRPX2 upon transfection with miR-192 and miR-215 in SW116 and HCT8 cells. SW116 and HCT8 cells were transfected miR-192 and miR-215 for 48 h, and then total protein was extracted for SPRX2 protein analysis by western blotting. ** P
    Figure Legend Snippet: MiR-192/miR-215 regulates SRPX2 expression in colon cancer cells. (A) miR-192 and miR-215 are the predicted miRNAs targeting SPRX2. (B) miR-192 and miR-215 expression increased in colon cancer cells. SW116 and HCT-8 cells were transfected miR-192 and miR-215 for 48 h, and then total RNA was extracted for real time RT-PCR. (C) Relative luciferase activity of the indicated SRPX2 reporter construct in SW116 cells. SW116 cells were co-transfected with SRPX2 promoter plasmid or miR-192 and miR-215 for 24 h. The cells were collected according to the protocol and analyzed. (D) miR-192 and miR-215 expression increased in colon cancer cells. SW116 and HCT8 cells were transfected miR-192 and miR-215 for 48 h, and then total RNA was extracted for SPRX2 mRNA analysis by real time RT-PCR. (E) Western blot assays were performed to detect the expression of SRPX2 upon transfection with miR-192 and miR-215 in SW116 and HCT8 cells. SW116 and HCT8 cells were transfected miR-192 and miR-215 for 48 h, and then total protein was extracted for SPRX2 protein analysis by western blotting. ** P

    Techniques Used: Expressing, Transfection, Quantitative RT-PCR, Luciferase, Activity Assay, Construct, Plasmid Preparation, Western Blot

    43) Product Images from "A MicroRNA196a2* and TP63 Circuit Regulated by Estrogen Receptor-? and ERK2 that Controls Breast Cancer Proliferation and Invasiveness Properties"

    Article Title: A MicroRNA196a2* and TP63 Circuit Regulated by Estrogen Receptor-? and ERK2 that Controls Breast Cancer Proliferation and Invasiveness Properties

    Journal: Hormones & cancer

    doi: 10.1007/s12672-012-0129-3

    Characterization of ERα, ERK2, and pSer5 RNA Pol II recruitment to the regulatory regions of miRNA genes
    Figure Legend Snippet: Characterization of ERα, ERK2, and pSer5 RNA Pol II recruitment to the regulatory regions of miRNA genes

    Techniques Used:

    44) Product Images from "Specific and Efficient Regression of Cancers Harboring KRAS Mutation by Targeted RNA Replacement"

    Article Title: Specific and Efficient Regression of Cancers Harboring KRAS Mutation by Targeted RNA Replacement

    Journal: Molecular Therapy

    doi: 10.1016/j.ymthe.2016.11.005

    Selective trans- Splicing Reaction and Target RNA Reduction by the Adenovirus Encoding for KRAS G12V RNA-Specific Ribozymes SW480 and HT-29 cells were infected with 20 and 50 MOI, respectively, of adenoviral vectors. (A) trans -spliced molecules (TSMs) generated in the cells were amplified, yielding a DNA fragment of 251 bp. The production of transgene RNA is shown by amplification of HSV-TK RNA (TK). HT-29 cells infected with a ribozyme-encoding adenoviral vector were mixed with mock-infected SW480 cells (mix). NTC denotes PCR control. Human GAPDH RNA was amplified as an internal control. (B) A representative sequence of TSMs generated from SW480 cells infected with Ad-3R1-TK or Ad-AR2-TK is shown. Arrows denote sequences around the splicing junction. (C) Target KRAS RNA was amplified in the cells infected with each adenoviral vector (left panel). The level of the target KRAS RNA in each cell was quantified using real-time PCR and expressed as a percentage of the level in mock-infected cells and as average with SD of three independent experiments (right panel).
    Figure Legend Snippet: Selective trans- Splicing Reaction and Target RNA Reduction by the Adenovirus Encoding for KRAS G12V RNA-Specific Ribozymes SW480 and HT-29 cells were infected with 20 and 50 MOI, respectively, of adenoviral vectors. (A) trans -spliced molecules (TSMs) generated in the cells were amplified, yielding a DNA fragment of 251 bp. The production of transgene RNA is shown by amplification of HSV-TK RNA (TK). HT-29 cells infected with a ribozyme-encoding adenoviral vector were mixed with mock-infected SW480 cells (mix). NTC denotes PCR control. Human GAPDH RNA was amplified as an internal control. (B) A representative sequence of TSMs generated from SW480 cells infected with Ad-3R1-TK or Ad-AR2-TK is shown. Arrows denote sequences around the splicing junction. (C) Target KRAS RNA was amplified in the cells infected with each adenoviral vector (left panel). The level of the target KRAS RNA in each cell was quantified using real-time PCR and expressed as a percentage of the level in mock-infected cells and as average with SD of three independent experiments (right panel).

    Techniques Used: Infection, Generated, Amplification, Plasmid Preparation, Polymerase Chain Reaction, Sequencing, Real-time Polymerase Chain Reaction

    Schematic Diagram of KRAS G12V RNA-Targeting trans -Splicing Ribozymes The target KRAS G12V RNA is represented with sequences around the splice site (italicized). Two trans -splicing ribozymes, KRib1 and KRib2, with extended IGS and antisense sequence against the target RNA are shown with the 3′ exon sequences capitalized. Potential base pairing between the ribozymes and the target transcript is indicated by vertical lines. The arrows indicate the 5′ and 3′ splicing sites of each ribozyme.
    Figure Legend Snippet: Schematic Diagram of KRAS G12V RNA-Targeting trans -Splicing Ribozymes The target KRAS G12V RNA is represented with sequences around the splice site (italicized). Two trans -splicing ribozymes, KRib1 and KRib2, with extended IGS and antisense sequence against the target RNA are shown with the 3′ exon sequences capitalized. Potential base pairing between the ribozymes and the target transcript is indicated by vertical lines. The arrows indicate the 5′ and 3′ splicing sites of each ribozyme.

    Techniques Used: Sequencing

    45) Product Images from "A multidimensional platform for the purification of non-coding RNA species"

    Article Title: A multidimensional platform for the purification of non-coding RNA species

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt668

    Separation of CCRF-SB and E. coli total RNA by SE-HPLC. ( A ) Typical profile of E. coli total RNA consisting of 23S, 16S rRNAs and co-eluting 5S rRNA and tRNA obtained on a Bio SEC-5 1000 Å column. ( B ) Typical profile of CCRF-SB total RNA consisting of 28S, 18S rRNAs and co-eluting 5.8S, 5S rRNA and tRNA obtained on a Bio SEC-5 1000 Å column. ( C ) Typical profile of E. coli total RNA consisting of 5S rRNA, tRNAs and co-eluting 16S and 23S rRNAs obtained on a Bio SEC-3 300 Å column. ( D ) Typical profile of CCRF-SB total RNA consisting of 5.8S, 5S rRNA, tRNAs and co-eluting 18S and 28S rRNAs obtained on a Bio SEC-3 300 Å column. The chromatograms show the analysis of 10 µg of total RNA extracted using Trizol reagent (Materials and Methods). ( E ) Separation of miRNA, tRNAs, 5.8S, 5S rRNAs, putative snRNAs and snoRNAs, and co-eluting 18S and 28S rRNAs from human lymphoblastic cell line CCRF-SB total RNA by IP RP HPLC obtained on a SOURCE 5RPC ST 4.5/150 column. The identity and purity of the RNAs collected in each fraction was validated with Bioanalyzer RNA 6000 Pico and Small RNA LabChips ( Supplementary Figures S1 and S3 ).
    Figure Legend Snippet: Separation of CCRF-SB and E. coli total RNA by SE-HPLC. ( A ) Typical profile of E. coli total RNA consisting of 23S, 16S rRNAs and co-eluting 5S rRNA and tRNA obtained on a Bio SEC-5 1000 Å column. ( B ) Typical profile of CCRF-SB total RNA consisting of 28S, 18S rRNAs and co-eluting 5.8S, 5S rRNA and tRNA obtained on a Bio SEC-5 1000 Å column. ( C ) Typical profile of E. coli total RNA consisting of 5S rRNA, tRNAs and co-eluting 16S and 23S rRNAs obtained on a Bio SEC-3 300 Å column. ( D ) Typical profile of CCRF-SB total RNA consisting of 5.8S, 5S rRNA, tRNAs and co-eluting 18S and 28S rRNAs obtained on a Bio SEC-3 300 Å column. The chromatograms show the analysis of 10 µg of total RNA extracted using Trizol reagent (Materials and Methods). ( E ) Separation of miRNA, tRNAs, 5.8S, 5S rRNAs, putative snRNAs and snoRNAs, and co-eluting 18S and 28S rRNAs from human lymphoblastic cell line CCRF-SB total RNA by IP RP HPLC obtained on a SOURCE 5RPC ST 4.5/150 column. The identity and purity of the RNAs collected in each fraction was validated with Bioanalyzer RNA 6000 Pico and Small RNA LabChips ( Supplementary Figures S1 and S3 ).

    Techniques Used: High Performance Liquid Chromatography, Size-exclusion Chromatography

    Reconstruction of the RNA landscape of Epstein-Barr virus-transformed TK6 cells by 2D SEC and IP RPC. ( A ) The 3D surface plot shows the analysis of 500 ng of total RNA extracted from TK6 cells using both 2D SEC (column 1: Bio SEC-3 300 Å; Column 2: Bio SEC-5 2000 Å) and IP RPC (SOURCE 5RPC ST 4.5/150). ( B ) Enhanced view of the smaller RNA species consisting of miRNA, tRNA, 5s and 5.8s rRNAs and putative snRNA and/or snoRNA. The identity and purity of the component RNA from orthographic projections of 2D SEC and IP RPC chromatograms is shown in Supplementary Figure S7 .
    Figure Legend Snippet: Reconstruction of the RNA landscape of Epstein-Barr virus-transformed TK6 cells by 2D SEC and IP RPC. ( A ) The 3D surface plot shows the analysis of 500 ng of total RNA extracted from TK6 cells using both 2D SEC (column 1: Bio SEC-3 300 Å; Column 2: Bio SEC-5 2000 Å) and IP RPC (SOURCE 5RPC ST 4.5/150). ( B ) Enhanced view of the smaller RNA species consisting of miRNA, tRNA, 5s and 5.8s rRNAs and putative snRNA and/or snoRNA. The identity and purity of the component RNA from orthographic projections of 2D SEC and IP RPC chromatograms is shown in Supplementary Figure S7 .

    Techniques Used: Transformation Assay, Size-exclusion Chromatography

    46) Product Images from "Pax6 associates with H3K4-specific histone methyltransferases Mll1, Mll2, and Set1a and regulates H3K4 methylation at promoters and enhancers"

    Article Title: Pax6 associates with H3K4-specific histone methyltransferases Mll1, Mll2, and Set1a and regulates H3K4 methylation at promoters and enhancers

    Journal: Epigenetics & Chromatin

    doi: 10.1186/s13072-016-0087-z

    Analysis of gene expression in Pax6 shRNA lens cell lines. a Knockdown of Pax6 by lentivirus shRNA (sh1 and sh2). Upper panel qRT-PCR. Lower panel western immunoblot. b Overlap of Pax6-bound genes and differentially expressed genes. Differentially expressed genes were detected by RNA-seq. c qRT-PCR validation of Pax6 positively regulated genes: Cap2, Farp1, Pax6 (see a ), Plekha1, Prox1, Tshz2, and Zfp536. p values
    Figure Legend Snippet: Analysis of gene expression in Pax6 shRNA lens cell lines. a Knockdown of Pax6 by lentivirus shRNA (sh1 and sh2). Upper panel qRT-PCR. Lower panel western immunoblot. b Overlap of Pax6-bound genes and differentially expressed genes. Differentially expressed genes were detected by RNA-seq. c qRT-PCR validation of Pax6 positively regulated genes: Cap2, Farp1, Pax6 (see a ), Plekha1, Prox1, Tshz2, and Zfp536. p values

    Techniques Used: Expressing, shRNA, Quantitative RT-PCR, Western Blot, RNA Sequencing Assay

    47) Product Images from "PIAS1 Regulates Breast Tumorigenesis through Selective Epigenetic Gene Silencing"

    Article Title: PIAS1 Regulates Breast Tumorigenesis through Selective Epigenetic Gene Silencing

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0089464

    PIAS1 regulates the expression of a panel of tumor suppressor genes. ( a ) Real-time quantitative PCR (Q-PCR) assay. MDA-MB231 cells containing control shRNA, PIAS1 shRNA1 or shRNA2 were cultured in DMEM plus 10% FBS (DMEM) or Stem Cell Media (SCM) for 30 h, and total RNA was used for Q-PCR assays with gene-specific primers. The gene names are labeled at the top left of each panel. The data were normalized by beta-Actin ( ACTB ) and presented as “Relative Expression” as compared to that in control shRNA cells under DMEM condition, which was set as “1” except for the ESR1 gene (the expression was not detectable in control shRNA cells). Shown is a representative of 3 independent experiments. Error bars represent SD. ND, not detected. See also Table S1 and Table S2 in File S1 . ( b ) Same as in a except that total RNA from fat pad tumor xenograft samples were used (n = 5). Error bars represent SEM. P values were determined by non-paired t -test.
    Figure Legend Snippet: PIAS1 regulates the expression of a panel of tumor suppressor genes. ( a ) Real-time quantitative PCR (Q-PCR) assay. MDA-MB231 cells containing control shRNA, PIAS1 shRNA1 or shRNA2 were cultured in DMEM plus 10% FBS (DMEM) or Stem Cell Media (SCM) for 30 h, and total RNA was used for Q-PCR assays with gene-specific primers. The gene names are labeled at the top left of each panel. The data were normalized by beta-Actin ( ACTB ) and presented as “Relative Expression” as compared to that in control shRNA cells under DMEM condition, which was set as “1” except for the ESR1 gene (the expression was not detectable in control shRNA cells). Shown is a representative of 3 independent experiments. Error bars represent SD. ND, not detected. See also Table S1 and Table S2 in File S1 . ( b ) Same as in a except that total RNA from fat pad tumor xenograft samples were used (n = 5). Error bars represent SEM. P values were determined by non-paired t -test.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Multiple Displacement Amplification, shRNA, Cell Culture, Labeling

    48) Product Images from "Efflux Transporter ArsK Is Responsible for Bacterial Resistance to Arsenite, Antimonite, Trivalent Roxarsone, and Methylarsenite"

    Article Title: Efflux Transporter ArsK Is Responsible for Bacterial Resistance to Arsenite, Antimonite, Trivalent Roxarsone, and Methylarsenite

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.01842-18

    ArsR2 negatively regulates the expression of arsK . (A) RT-PCR analysis illustrating the cotranscription of arsR2 and arsK . Total RNA was extracted from strain GW4 grown in MMNH 4 medium with 1 mM As(III). M, molecular weight marker (DL 2000 plus). In lanes 1 and 4, total DNA was used as the template; in lanes 2 and 5, total RNA was used as the template; in lines 3 and 6, double-distilled water (ddH 2 O) was used as the template. The horizontal lines represent the locations of primers. (B) EMSAs. FAM-labeled arsR2 - arsK probe interacted with ArsR2 protein. The amounts of DNA probes and ArsR2 are shown in the tables above each panel. Competition EMSAs including arsR2 - arsK probe and ArsR2 protein with increasing amounts of As(III) (C), Sb(III) (D), Rox(III) (E), MAs(III) (F), As(V) (G), or dimethyl-As(III) (H). The amount of DNA probe was 100 ng, the amount of ArsR2 was 1.0 μM, and the amounts of each metalloid are shown in the tables above each panel.
    Figure Legend Snippet: ArsR2 negatively regulates the expression of arsK . (A) RT-PCR analysis illustrating the cotranscription of arsR2 and arsK . Total RNA was extracted from strain GW4 grown in MMNH 4 medium with 1 mM As(III). M, molecular weight marker (DL 2000 plus). In lanes 1 and 4, total DNA was used as the template; in lanes 2 and 5, total RNA was used as the template; in lines 3 and 6, double-distilled water (ddH 2 O) was used as the template. The horizontal lines represent the locations of primers. (B) EMSAs. FAM-labeled arsR2 - arsK probe interacted with ArsR2 protein. The amounts of DNA probes and ArsR2 are shown in the tables above each panel. Competition EMSAs including arsR2 - arsK probe and ArsR2 protein with increasing amounts of As(III) (C), Sb(III) (D), Rox(III) (E), MAs(III) (F), As(V) (G), or dimethyl-As(III) (H). The amount of DNA probe was 100 ng, the amount of ArsR2 was 1.0 μM, and the amounts of each metalloid are shown in the tables above each panel.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Molecular Weight, Marker, Labeling

    49) Product Images from "Nitrilase and Fhit homologs are encoded as fusion proteins in Drosophila melanogaster and Caenorhabditis elegans"

    Article Title: Nitrilase and Fhit homologs are encoded as fusion proteins in Drosophila melanogaster and Caenorhabditis elegans

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    Analysis of alternative transcripts of human NIT1 by RT-PCR. RT-PCR of HeLa RNA was performed with primers in different exons. Lanes 1–6: exons 1 and 3 (transcript 2); exons 1C and 3 (transcript 5); exons 1A and 3 (transcripts 3, upper band and 4, lower band); exons 2 and 3 (transcripts 2–4); exons 1 and 1C (transcript 5); and exons 1 and 2 (transcript 2).
    Figure Legend Snippet: Analysis of alternative transcripts of human NIT1 by RT-PCR. RT-PCR of HeLa RNA was performed with primers in different exons. Lanes 1–6: exons 1 and 3 (transcript 2); exons 1C and 3 (transcript 5); exons 1A and 3 (transcripts 3, upper band and 4, lower band); exons 2 and 3 (transcripts 2–4); exons 1 and 1C (transcript 5); and exons 1 and 2 (transcript 2).

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    50) Product Images from "Association of a single nucleotide polymorphism in the 5' upstream region of the porcine myosin heavy chain 4 gene with meat quality traits in pigs) Association of a single nucleotide polymorphism in the 5' upstream region of the porcine myosin heavy chain 4 gene with meat quality traits in pigs"

    Article Title: Association of a single nucleotide polymorphism in the 5' upstream region of the porcine myosin heavy chain 4 gene with meat quality traits in pigs) Association of a single nucleotide polymorphism in the 5' upstream region of the porcine myosin heavy chain 4 gene with meat quality traits in pigs

    Journal: Animal Science Journal = Nihon Chikusan Gakkaiho

    doi: 10.1111/asj.12442

    Comparison of messenger RNA (mRNA) expression levels of the porcine MYH4 gene on g.‐1398G > T site. Quantitative real‐time PCR analysis was used to determine the expression levels of MYH4 mRNA in the Longissimus dorsi muscle of each indicated genotyped Landrace pig. The mRNAs of MYH4 were normalized to those of GAPDH and compared between different genotype. All values were described with mean ± standard error of the mean from three independent experiments. Different superscripts (a or b) above the error bar show significantly different genotypes on the single nucleotide polymorphism g.‐1398 site ( P
    Figure Legend Snippet: Comparison of messenger RNA (mRNA) expression levels of the porcine MYH4 gene on g.‐1398G > T site. Quantitative real‐time PCR analysis was used to determine the expression levels of MYH4 mRNA in the Longissimus dorsi muscle of each indicated genotyped Landrace pig. The mRNAs of MYH4 were normalized to those of GAPDH and compared between different genotype. All values were described with mean ± standard error of the mean from three independent experiments. Different superscripts (a or b) above the error bar show significantly different genotypes on the single nucleotide polymorphism g.‐1398 site ( P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    51) Product Images from "Long noncoding RNA TP73-AS1 accelerates the epithelial ovarian cancer via epigenetically repressing p21"

    Article Title: Long noncoding RNA TP73-AS1 accelerates the epithelial ovarian cancer via epigenetically repressing p21

    Journal: American Journal of Translational Research

    doi:

    TP73-AS1 epigenetically inhibits p21 through binding with EZH2. A. The subcellular location of TP73-AS1 in the nuclear and the cytoplasm fraction. B. The level of CDK inhibitors (CKIs), including p15, p16, p21, p27, p57. C. Western blot revealed the p21 protein in the TP73-AS1 silencing transfection. D. RNA-binding protein immunoprecipitation (RIP) assay showed the binding of TP73-AS1 with EZH2. E. Chromatin immunoprecipitation (ChIP) revealed the direct bound of EZH2 with p21 promoter region. F. Kaplan-Meier survival curve analysis based on the TCGA database suggested the prognosis of EOC patients with higher or lower group. **
    Figure Legend Snippet: TP73-AS1 epigenetically inhibits p21 through binding with EZH2. A. The subcellular location of TP73-AS1 in the nuclear and the cytoplasm fraction. B. The level of CDK inhibitors (CKIs), including p15, p16, p21, p27, p57. C. Western blot revealed the p21 protein in the TP73-AS1 silencing transfection. D. RNA-binding protein immunoprecipitation (RIP) assay showed the binding of TP73-AS1 with EZH2. E. Chromatin immunoprecipitation (ChIP) revealed the direct bound of EZH2 with p21 promoter region. F. Kaplan-Meier survival curve analysis based on the TCGA database suggested the prognosis of EOC patients with higher or lower group. **

    Techniques Used: Binding Assay, Western Blot, Transfection, RNA Binding Assay, Immunoprecipitation, Chromatin Immunoprecipitation

    52) Product Images from "Epigallocatechin-3-Gallate Ameliorates Experimental Autoimmune Encephalomyelitis by Altering Balance among CD4+ T-Cell Subsets"

    Article Title: Epigallocatechin-3-Gallate Ameliorates Experimental Autoimmune Encephalomyelitis by Altering Balance among CD4+ T-Cell Subsets

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2011.09.007

    Dietary EGCG supplementation reduces circulating ICAM-1 and down-regulates CCR6 expression in CD4 + T cells but does not affect CCL20 expression in the CNS. On day 12 after immunization, mice were euthanized and blood, spleens, brains, and spinal cords were collected. A: Blood was centrifuged to obtain plasma, and sICAM-1 levels in the plasma samples were determined by ELISA. B and C: Spleen cells were isolated and CCR6 expression in CD4 + T cells was determined by flow cytometry. These data are expressed as percentage of CCR6 + CD4 + T cells ( B ) and CCR6 expression level per cell, as indicated by mean fluorescence intensity (MFI; C ). D: Total RNA extracted from the CNS tissue (brains and spinal cords) was used to determine CCL20 mRNA expression using real-time RT-PCR. All values in this figure are mean ± SEM ( n = 6/group). Significant differences were determined by the nonpaired Student's t -test. * P
    Figure Legend Snippet: Dietary EGCG supplementation reduces circulating ICAM-1 and down-regulates CCR6 expression in CD4 + T cells but does not affect CCL20 expression in the CNS. On day 12 after immunization, mice were euthanized and blood, spleens, brains, and spinal cords were collected. A: Blood was centrifuged to obtain plasma, and sICAM-1 levels in the plasma samples were determined by ELISA. B and C: Spleen cells were isolated and CCR6 expression in CD4 + T cells was determined by flow cytometry. These data are expressed as percentage of CCR6 + CD4 + T cells ( B ) and CCR6 expression level per cell, as indicated by mean fluorescence intensity (MFI; C ). D: Total RNA extracted from the CNS tissue (brains and spinal cords) was used to determine CCL20 mRNA expression using real-time RT-PCR. All values in this figure are mean ± SEM ( n = 6/group). Significant differences were determined by the nonpaired Student's t -test. * P

    Techniques Used: Expressing, Mouse Assay, Enzyme-linked Immunosorbent Assay, Isolation, Flow Cytometry, Cytometry, Fluorescence, Quantitative RT-PCR

    Dietary EGCG suppresses T-bet and RORγt expression. On day 12 after immunization, mice were euthanized and spleens, brains, and spinal cords were collected. A: Isolated spleen cells were used to conduct intracellular staining for T-bet and RORγt in CD4 + T cells and analyzed by flow cytometry. B: Total RNA extracted from the CNS tissue (brains and spinal cords) was used to determine T-bet and RORγt mRNA expression using real-time RT-PCR. All values in this figure are mean ± SEM ( n = 6/group). Significant differences were determined by the nonpaired Student's t -test. * P
    Figure Legend Snippet: Dietary EGCG suppresses T-bet and RORγt expression. On day 12 after immunization, mice were euthanized and spleens, brains, and spinal cords were collected. A: Isolated spleen cells were used to conduct intracellular staining for T-bet and RORγt in CD4 + T cells and analyzed by flow cytometry. B: Total RNA extracted from the CNS tissue (brains and spinal cords) was used to determine T-bet and RORγt mRNA expression using real-time RT-PCR. All values in this figure are mean ± SEM ( n = 6/group). Significant differences were determined by the nonpaired Student's t -test. * P

    Techniques Used: Expressing, Mouse Assay, Isolation, Staining, Flow Cytometry, Cytometry, Quantitative RT-PCR

    Dietary EGCG inhibits Th1 and Th17 response in peripheral lymphoid organs of mice with EAE. On day 12 after immunization, mice were euthanized and spleen and draining LNs were collected. Spleen and LN cells were stimulated in vitro with 100 μg/mL MOG 35-55 peptide for 72 hours to determine secretion of IFN-γ and IL-17 using ELISA ( A ). In addition, these cells were restimulated with PMA and ionomycin in the presence of Golgi Stop for an additional 4 hours for analysis of intracellular levels of IFN-γ and IL-17 to identify Th1 and Th17 populations in CD4 + T cells of spleen ( B ) and LNs ( C ). D: IL-12/IL-23 total p40 levels in plasma samples were determined by ELISA. Total RNA extracted from the splenocytes was used to determine IL-12p35, IL-12p40, and IL-23p19 mRNA expression levels using real-time RT-PCR. All values in this figure are mean ± SEM ( n = 8/group). Significant differences were determined by the nonpaired Student's t -test. * P
    Figure Legend Snippet: Dietary EGCG inhibits Th1 and Th17 response in peripheral lymphoid organs of mice with EAE. On day 12 after immunization, mice were euthanized and spleen and draining LNs were collected. Spleen and LN cells were stimulated in vitro with 100 μg/mL MOG 35-55 peptide for 72 hours to determine secretion of IFN-γ and IL-17 using ELISA ( A ). In addition, these cells were restimulated with PMA and ionomycin in the presence of Golgi Stop for an additional 4 hours for analysis of intracellular levels of IFN-γ and IL-17 to identify Th1 and Th17 populations in CD4 + T cells of spleen ( B ) and LNs ( C ). D: IL-12/IL-23 total p40 levels in plasma samples were determined by ELISA. Total RNA extracted from the splenocytes was used to determine IL-12p35, IL-12p40, and IL-23p19 mRNA expression levels using real-time RT-PCR. All values in this figure are mean ± SEM ( n = 8/group). Significant differences were determined by the nonpaired Student's t -test. * P

    Techniques Used: Mouse Assay, In Vitro, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR

    53) Product Images from "Differentially expressed, variant U1 snRNAs regulate gene expression in human cells"

    Article Title: Differentially expressed, variant U1 snRNAs regulate gene expression in human cells

    Journal: Genome Research

    doi: 10.1101/gr.142968.112

    vU1 snRNAs are packaged into RNP complexes. qRT-PCR analysis of U1 snRNA and vU1 snRNA levels in HeLa ( A ) and hESC ( B ) extracts, before and after immunoprecipitation with anti-Sm (Y-12) antibody. The non-Sm-containing 7SK RNA level was used as a negative
    Figure Legend Snippet: vU1 snRNAs are packaged into RNP complexes. qRT-PCR analysis of U1 snRNA and vU1 snRNA levels in HeLa ( A ) and hESC ( B ) extracts, before and after immunoprecipitation with anti-Sm (Y-12) antibody. The non-Sm-containing 7SK RNA level was used as a negative

    Techniques Used: Quantitative RT-PCR, Immunoprecipitation

    54) Product Images from "Differentially expressed, variant U1 snRNAs regulate gene expression in human cells"

    Article Title: Differentially expressed, variant U1 snRNAs regulate gene expression in human cells

    Journal: Genome Research

    doi: 10.1101/gr.142968.112

    vU1 snRNAs are packaged into RNP complexes. qRT-PCR analysis of U1 snRNA and vU1 snRNA levels in HeLa ( A ) and hESC ( B ) extracts, before and after immunoprecipitation with anti-Sm (Y-12) antibody. The non-Sm-containing 7SK RNA level was used as a negative
    Figure Legend Snippet: vU1 snRNAs are packaged into RNP complexes. qRT-PCR analysis of U1 snRNA and vU1 snRNA levels in HeLa ( A ) and hESC ( B ) extracts, before and after immunoprecipitation with anti-Sm (Y-12) antibody. The non-Sm-containing 7SK RNA level was used as a negative

    Techniques Used: Quantitative RT-PCR, Immunoprecipitation

    55) Product Images from "Gene Expression Profiling Associated with Angiotensin II Type 2 Receptor-Induced Apoptosis in Human Prostate Cancer Cells"

    Article Title: Gene Expression Profiling Associated with Angiotensin II Type 2 Receptor-Induced Apoptosis in Human Prostate Cancer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0092253

    The expression of Bcl-2 in transduced DU145 cells. DU145 cells were transduced with either Ad-G-AT2R-EGFP(AT2R) or Ad-CMV-EGFP (EGFP) for 2d at 200 ifu/cell. These treatments were followed by total RNA isolation and then real-time RT-PCR analysis using specific oligonucleotide primers and Taqman probes. All data were normalized against levels of GAPDH mRNA expression within the same sample. Columns, mean from three separate experiments.
    Figure Legend Snippet: The expression of Bcl-2 in transduced DU145 cells. DU145 cells were transduced with either Ad-G-AT2R-EGFP(AT2R) or Ad-CMV-EGFP (EGFP) for 2d at 200 ifu/cell. These treatments were followed by total RNA isolation and then real-time RT-PCR analysis using specific oligonucleotide primers and Taqman probes. All data were normalized against levels of GAPDH mRNA expression within the same sample. Columns, mean from three separate experiments.

    Techniques Used: Expressing, Transduction, Isolation, Quantitative RT-PCR

    Viral vector–mediated expression of AT2R in prostate cancer cell lines. DU145 cells were transduced with Ad-G-AT2R-EGFP(B and D) and Ad-CMV-EGFP (A and C) (100 ifu/cell) for 2 days, and cell morphology was examined under a fluorescence microscope. Scale bars, 50μm. Total RNA was extracted from transduced DU145 cells and AT2Rs were detected by real time RT-PCR. Ethidium bromide–stained gels show AT2R (E) and GAPDH (F) transcripts in transduced cells. M, DL 2,000 DNA Maker (Takara); 1, 3, 5, 7, 9: transduced with 10, 20, 50, 100, 200 ifu/cell separately of Ad-G-AT2R-EGFP; 2, 4, 6, 8, 10: transduced with 10, 20, 50, 100, 200 ifu/cell separately of Ad-CMV-EGFP.
    Figure Legend Snippet: Viral vector–mediated expression of AT2R in prostate cancer cell lines. DU145 cells were transduced with Ad-G-AT2R-EGFP(B and D) and Ad-CMV-EGFP (A and C) (100 ifu/cell) for 2 days, and cell morphology was examined under a fluorescence microscope. Scale bars, 50μm. Total RNA was extracted from transduced DU145 cells and AT2Rs were detected by real time RT-PCR. Ethidium bromide–stained gels show AT2R (E) and GAPDH (F) transcripts in transduced cells. M, DL 2,000 DNA Maker (Takara); 1, 3, 5, 7, 9: transduced with 10, 20, 50, 100, 200 ifu/cell separately of Ad-G-AT2R-EGFP; 2, 4, 6, 8, 10: transduced with 10, 20, 50, 100, 200 ifu/cell separately of Ad-CMV-EGFP.

    Techniques Used: Plasmid Preparation, Expressing, Transduction, Fluorescence, Microscopy, Quantitative RT-PCR, Staining

    56) Product Images from "Transcriptome-wide analysis of compression-induced microRNA expression alteration in breast cancer for mining therapeutic targets"

    Article Title: Transcriptome-wide analysis of compression-induced microRNA expression alteration in breast cancer for mining therapeutic targets

    Journal: Oncotarget

    doi: 10.18632/oncotarget.8322

    Analysis of fold change in expression level of putative target mRNAs of compression-upregulated microRNAs Compression-induced decrease in expression levels of microRNA target genes in A. BT-474, B. MCF7, C. SK-BR-3, D. MDA-MB-231, E. CAF1, F. CAF2, G. CAF3, and H. CAF4 cells. Compression-induced target-gene downregulation was analyzed in the same samples of total RNA used for microRNA array analysis. Target mRNA downregulation showing more than 5-fold change on average was presented for analysis.
    Figure Legend Snippet: Analysis of fold change in expression level of putative target mRNAs of compression-upregulated microRNAs Compression-induced decrease in expression levels of microRNA target genes in A. BT-474, B. MCF7, C. SK-BR-3, D. MDA-MB-231, E. CAF1, F. CAF2, G. CAF3, and H. CAF4 cells. Compression-induced target-gene downregulation was analyzed in the same samples of total RNA used for microRNA array analysis. Target mRNA downregulation showing more than 5-fold change on average was presented for analysis.

    Techniques Used: Expressing, Multiple Displacement Amplification

    57) Product Images from "Anti-obesity effects of Lysimachia foenum-graecum characterized by decreased adipogenesis and regulated lipid metabolism"

    Article Title: Anti-obesity effects of Lysimachia foenum-graecum characterized by decreased adipogenesis and regulated lipid metabolism

    Journal: Experimental & Molecular Medicine

    doi: 10.3858/emm.2011.43.4.025

    LFE modulates the expression of genes involved in lipid metabolism. Differentiated 3T3-L1 adipocytes (A) or C2C12 cells (B) were treated with 10 µg/ml LFE for 24 h. Total RNA was isolated and analyzed by qPCR for the expression of lipogenic genes
    Figure Legend Snippet: LFE modulates the expression of genes involved in lipid metabolism. Differentiated 3T3-L1 adipocytes (A) or C2C12 cells (B) were treated with 10 µg/ml LFE for 24 h. Total RNA was isolated and analyzed by qPCR for the expression of lipogenic genes

    Techniques Used: Expressing, Isolation, Real-time Polymerase Chain Reaction

    58) Product Images from "ZNF224, Krüppel like zinc finger protein, induces cell growth and apoptosis-resistance by down-regulation of p21 and p53 via miR-663a"

    Article Title: ZNF224, Krüppel like zinc finger protein, induces cell growth and apoptosis-resistance by down-regulation of p21 and p53 via miR-663a

    Journal: Oncotarget

    doi: 10.18632/oncotarget.8870

    ZNF224 binds to miR-663a promoter A. miR-663a promoter sequence analysis. Two regions containing 5′-CAGC-3′ sequence were found within - 500 bp. Arrow indicates primer binding sites for PCR. B and C. FLAG-ZNF224 was transfected to HEK293 cells, and ChIP assay using normal or FLAG IgG was carried out. PCR was performed using miR-663a promoter primers between -350 bp and -89 bp. In addition, ChIPed DNA was analyzed with qPCR. Data are from at least three independent experiments (n=3). D. FLAG-ZNF224 (0.5 1, and 2 μg) was transfected to MCF-7 cells in a dose-dependent manner for 24 h, and miR-663a transcript level was examined by qRT-PCR. miR-663a transcript level was normalized to U6 RNA. Data are from at least three independent experiments (n=3).
    Figure Legend Snippet: ZNF224 binds to miR-663a promoter A. miR-663a promoter sequence analysis. Two regions containing 5′-CAGC-3′ sequence were found within - 500 bp. Arrow indicates primer binding sites for PCR. B and C. FLAG-ZNF224 was transfected to HEK293 cells, and ChIP assay using normal or FLAG IgG was carried out. PCR was performed using miR-663a promoter primers between -350 bp and -89 bp. In addition, ChIPed DNA was analyzed with qPCR. Data are from at least three independent experiments (n=3). D. FLAG-ZNF224 (0.5 1, and 2 μg) was transfected to MCF-7 cells in a dose-dependent manner for 24 h, and miR-663a transcript level was examined by qRT-PCR. miR-663a transcript level was normalized to U6 RNA. Data are from at least three independent experiments (n=3).

    Techniques Used: Sequencing, Binding Assay, Polymerase Chain Reaction, Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    ZNF224 controls cell growth by regulating expressions of p21 and p53 via miR-663a A and B. MCF-7 cells were transfected with LNA-ctrl or miR-663a (20 nM) in the presence or absence of miR-663a antagonist (40 nM) as indicated. The expression of p21 and p53 was examined by qRT-PCR and immunoblot, respectively. GAPDH and tubulin were used as loading control in qRT-PCR and immunoblot, respectively. Data are from at least three independent experiments (n=3). C and D . In addition, colony forming ability was analyzed (*, vs. LNA-ctrl; **, vs. miR-663a) and subjected to iCelligence for the growth analysis in the presence or absence of CPT (0.1 μM). E and F. MCF-7 cells were transfected with FLAG-ZNF224 (2 μg) in the presence or absence of miR-663a antagonist (40 nM). The expression of p21, p53, miR-663a, and ZNF224 was examined by qRT-PCR and immunoblot, respectively. GAPDH and U6 RNA were used as loading control for mRNA and microRNA, respectively, in qRT-PCR, and tubulin was used as a loading control for immunoblot. Data are from at least three independent experiments (n=3). G and H . In addition, colony forming ability was analyzed (#, vs. EV; †, vs. ZNF224) and subjected to iCelligence for the growth analysis in the presence or absence of CPT (0.1 μM). I and J . MCF-7 cells were transfected with control or ZNF224 si-RNA (20 nM), and the expression of p21, p53, miR-663a, and ZNF224 was examined by qRT-PCR and immunoblot, respectively (**, vs. control si-RNA). GAPDH and U6 RNA were used as loading control for mRNA and microRNA, respectively, in qRT-PCR, and tubulin was used as a loading control for immunoblot. Data represent the mean ± SEM of three independent experiments. * P
    Figure Legend Snippet: ZNF224 controls cell growth by regulating expressions of p21 and p53 via miR-663a A and B. MCF-7 cells were transfected with LNA-ctrl or miR-663a (20 nM) in the presence or absence of miR-663a antagonist (40 nM) as indicated. The expression of p21 and p53 was examined by qRT-PCR and immunoblot, respectively. GAPDH and tubulin were used as loading control in qRT-PCR and immunoblot, respectively. Data are from at least three independent experiments (n=3). C and D . In addition, colony forming ability was analyzed (*, vs. LNA-ctrl; **, vs. miR-663a) and subjected to iCelligence for the growth analysis in the presence or absence of CPT (0.1 μM). E and F. MCF-7 cells were transfected with FLAG-ZNF224 (2 μg) in the presence or absence of miR-663a antagonist (40 nM). The expression of p21, p53, miR-663a, and ZNF224 was examined by qRT-PCR and immunoblot, respectively. GAPDH and U6 RNA were used as loading control for mRNA and microRNA, respectively, in qRT-PCR, and tubulin was used as a loading control for immunoblot. Data are from at least three independent experiments (n=3). G and H . In addition, colony forming ability was analyzed (#, vs. EV; †, vs. ZNF224) and subjected to iCelligence for the growth analysis in the presence or absence of CPT (0.1 μM). I and J . MCF-7 cells were transfected with control or ZNF224 si-RNA (20 nM), and the expression of p21, p53, miR-663a, and ZNF224 was examined by qRT-PCR and immunoblot, respectively (**, vs. control si-RNA). GAPDH and U6 RNA were used as loading control for mRNA and microRNA, respectively, in qRT-PCR, and tubulin was used as a loading control for immunoblot. Data represent the mean ± SEM of three independent experiments. * P

    Techniques Used: Transfection, Expressing, Quantitative RT-PCR, Cycling Probe Technology

    ZNF224 increases colony forming ability of MCF-7 cells A. MCF-7 cells were transfected with empty vector (E.V) or FLAG-ZNF224 (2 μg) and subjected to colony forming assay for 14 days (*, vs. E.V). B. The over-expression of FLAG-ZNF224 was confirmed by RT-PCR and immunoblot. C. MCF-7 cells were transfected with control or ZNF224 targeting si-RNA (20 nM), and colony forming ability was examined (**, vs. si-control). D. The knock-down of ZNF224 was examined by RT-PCR and immunoblot. GAPDH and tubulin were used as loading control. Data represent the mean ± SEM of three independent experiments. * P
    Figure Legend Snippet: ZNF224 increases colony forming ability of MCF-7 cells A. MCF-7 cells were transfected with empty vector (E.V) or FLAG-ZNF224 (2 μg) and subjected to colony forming assay for 14 days (*, vs. E.V). B. The over-expression of FLAG-ZNF224 was confirmed by RT-PCR and immunoblot. C. MCF-7 cells were transfected with control or ZNF224 targeting si-RNA (20 nM), and colony forming ability was examined (**, vs. si-control). D. The knock-down of ZNF224 was examined by RT-PCR and immunoblot. GAPDH and tubulin were used as loading control. Data represent the mean ± SEM of three independent experiments. * P

    Techniques Used: Transfection, Plasmid Preparation, Over Expression, Reverse Transcription Polymerase Chain Reaction

    59) Product Images from "Mice with cardiomyocyte-specific disruption of the endothelin-1 gene are resistant to hyperthyroid cardiac hypertrophy"

    Article Title: Mice with cardiomyocyte-specific disruption of the endothelin-1 gene are resistant to hyperthyroid cardiac hypertrophy

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0307159101

    Quantitative RT-PCR analysis of ET-1 mRNA in cardiac myocytes. ( A ) Autoradiogram of a Southern blot of ET-1 exon 2 amplicons from cardiomyocyte preparations of ET-1 flox/flox ; Cre + and ET- 1 flox/flox ; Cre - mice with and without 24 h of treatment with T3 in vivo . ( B ) Quantitation of radioactive signal from the gel in A (by PhosphorImager). ( C ) Analysis of the same RNA preparations assessed by real-time RT-PCR. Cardiac myocytes from two animals were independently assessed, and the mean value is provided.
    Figure Legend Snippet: Quantitative RT-PCR analysis of ET-1 mRNA in cardiac myocytes. ( A ) Autoradiogram of a Southern blot of ET-1 exon 2 amplicons from cardiomyocyte preparations of ET-1 flox/flox ; Cre + and ET- 1 flox/flox ; Cre - mice with and without 24 h of treatment with T3 in vivo . ( B ) Quantitation of radioactive signal from the gel in A (by PhosphorImager). ( C ) Analysis of the same RNA preparations assessed by real-time RT-PCR. Cardiac myocytes from two animals were independently assessed, and the mean value is provided.

    Techniques Used: Quantitative RT-PCR, Southern Blot, Mouse Assay, In Vivo, Quantitation Assay

    60) Product Images from "Evolutionary analysis reveals regulatory and functional landscape of coding and non-coding RNA editingThe Landscape of A-to-I RNA Editome Is Shaped by Both Positive and Purifying Selection"

    Article Title: Evolutionary analysis reveals regulatory and functional landscape of coding and non-coding RNA editingThe Landscape of A-to-I RNA Editome Is Shaped by Both Positive and Purifying Selection

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1006563

    Cis regulatory features explain and predict the divergence of RNA editing levels. ( A ) The difference in ADAR binding motif scores between sites that are edited (≥10%) in D . mel but not edited (≤1.5%) in other species (green) compared to the difference in sites that are edited (≥10%) in both species (purple). *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001 (one-tailed Mann-Whitney U test) ( B ) An example of the relationship between structure and editing level changes across species. The editing site is indicated by the purple arrows. ( C ) The difference in free energy, stem length, and paired bases between sites that are edited (≥10%) in D . mel but not edited (≤1.5%) in other species and control sites that are edited (≥10%) in both species. The numbers of editing sites are indicated above the bars. *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001 (one-tailed Mann-Whitney U test) ( D ) Relative importance of ADAR motif and dsRNA structural features to predict the establishment of editing (left) and the variation in editing levels (right). In the Presence/Absence editing scenario (left), given that a site was edited (≥10%) in one species, we predicted whether sites were edited (≥10%) or not edited (≤1.5%) in another species. In the Presence/Presence editing scenario (right), given the editing level in one species, we predicted the editing level in another species, requiring that the sites were edited (≥10%) in both species. The D . mel — D . vir comparison is shown. Features with positive and negative relationships with presence of editing (left) or increase of editing level (right) are marked in red and black, respectively. ( E ) Comparison of predictive accuracy of editing levels of two models (with or without motif and structural features). Given the editing levels in D . mel , editing levels in each of the other species were predicted using the random forests model. Sites with editing level ≥10% in at least one of the paired species were included in the analysis.
    Figure Legend Snippet: Cis regulatory features explain and predict the divergence of RNA editing levels. ( A ) The difference in ADAR binding motif scores between sites that are edited (≥10%) in D . mel but not edited (≤1.5%) in other species (green) compared to the difference in sites that are edited (≥10%) in both species (purple). *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001 (one-tailed Mann-Whitney U test) ( B ) An example of the relationship between structure and editing level changes across species. The editing site is indicated by the purple arrows. ( C ) The difference in free energy, stem length, and paired bases between sites that are edited (≥10%) in D . mel but not edited (≤1.5%) in other species and control sites that are edited (≥10%) in both species. The numbers of editing sites are indicated above the bars. *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001 (one-tailed Mann-Whitney U test) ( D ) Relative importance of ADAR motif and dsRNA structural features to predict the establishment of editing (left) and the variation in editing levels (right). In the Presence/Absence editing scenario (left), given that a site was edited (≥10%) in one species, we predicted whether sites were edited (≥10%) or not edited (≤1.5%) in another species. In the Presence/Presence editing scenario (right), given the editing level in one species, we predicted the editing level in another species, requiring that the sites were edited (≥10%) in both species. The D . mel — D . vir comparison is shown. Features with positive and negative relationships with presence of editing (left) or increase of editing level (right) are marked in red and black, respectively. ( E ) Comparison of predictive accuracy of editing levels of two models (with or without motif and structural features). Given the editing levels in D . mel , editing levels in each of the other species were predicted using the random forests model. Sites with editing level ≥10% in at least one of the paired species were included in the analysis.

    Techniques Used: Binding Assay, One-tailed Test, MANN-WHITNEY

    61) Product Images from "Primary Human Placental Trophoblasts are Permissive for Zika Virus (ZIKV) Replication"

    Article Title: Primary Human Placental Trophoblasts are Permissive for Zika Virus (ZIKV) Replication

    Journal: Scientific Reports

    doi: 10.1038/srep41389

    Significant decrease in the level of the TLR7/8 ligand binding miR-21 miRNA, but not C19MC miRNA species following ZIKV infection in primary human trophoblasts. Exosomal RNA was isolated from primary trophoblasts of ten donors following ZIKV infection with 1 × 10 5 viral copies, or mock infected controls. RNA was then isolated from trophoblast cultures 3–5 days post infection. TaqMan qPCR assays were employed for species-specific miRNA quantification. Significant differences in miRNAs were observed for miR-21 (decreased ~1.5 fold (0.68 ± 0.2 SD), p = 0.001), while transcripts of the C19 miRNA cluster were not significantly changed by ZIKV infection. Fold change in miRNA species were calculated by the delta delta Ct method, normalizing first to U6 and then mean delta Ct of mock infected controls. Data was filtered for outliers as designated by a Q of 0.01. Significance was determined using t-tests, with designation of significance annotated by c (c significance p = 0.001).
    Figure Legend Snippet: Significant decrease in the level of the TLR7/8 ligand binding miR-21 miRNA, but not C19MC miRNA species following ZIKV infection in primary human trophoblasts. Exosomal RNA was isolated from primary trophoblasts of ten donors following ZIKV infection with 1 × 10 5 viral copies, or mock infected controls. RNA was then isolated from trophoblast cultures 3–5 days post infection. TaqMan qPCR assays were employed for species-specific miRNA quantification. Significant differences in miRNAs were observed for miR-21 (decreased ~1.5 fold (0.68 ± 0.2 SD), p = 0.001), while transcripts of the C19 miRNA cluster were not significantly changed by ZIKV infection. Fold change in miRNA species were calculated by the delta delta Ct method, normalizing first to U6 and then mean delta Ct of mock infected controls. Data was filtered for outliers as designated by a Q of 0.01. Significance was determined using t-tests, with designation of significance annotated by c (c significance p = 0.001).

    Techniques Used: Ligand Binding Assay, Infection, Isolation, Real-time Polymerase Chain Reaction

    62) Product Images from "Interleukin-6 (IL-6) receptor/IL-6 fusion protein (Hyper IL-6) effects on the neonatal mouse brain: possible role for IL-6 trans-signaling in brain development and functional neurobehavioral outcomes"

    Article Title: Interleukin-6 (IL-6) receptor/IL-6 fusion protein (Hyper IL-6) effects on the neonatal mouse brain: possible role for IL-6 trans-signaling in brain development and functional neurobehavioral outcomes

    Journal: Brain, behavior, and immunity

    doi: 10.1016/j.bbi.2012.08.017

    RNase protection assay for (A) acute response genes and inflammatory cytokines ICAM-1, iNOS, A20, MAC-1 and EB22 (B) Interleukin 1 related genes and (C) TNF and IL-6 related genes on total RNA isolated from cortex at PND5 and 11 from mice injected with
    Figure Legend Snippet: RNase protection assay for (A) acute response genes and inflammatory cytokines ICAM-1, iNOS, A20, MAC-1 and EB22 (B) Interleukin 1 related genes and (C) TNF and IL-6 related genes on total RNA isolated from cortex at PND5 and 11 from mice injected with

    Techniques Used: Rnase Protection Assay, Isolation, Mouse Assay, Injection

    63) Product Images from "The Krüppel-like factor 9 cistrome in mouse hippocampal neurons reveals predominant transcriptional repression via proximal promoter binding"

    Article Title: The Krüppel-like factor 9 cistrome in mouse hippocampal neurons reveals predominant transcriptional repression via proximal promoter binding

    Journal: BMC Genomics

    doi: 10.1186/s12864-017-3640-7

    Identification of Klf9-regulated genes in HT22 cells by RNA-sequencing. a Treatment of HT22 [TR/TO-Klf9] cells with doxycycline (dox; 1 μg/ml) for 8 h increased Klf9 mRNA ~10 fold compared to vehicle treated cells, but had no effect in parent HT22 cells. The baseline Klf9 mRNA level did not differ between parent and [TR/TO-Klf9] line. Means with the same letter are not significantly different ( p
    Figure Legend Snippet: Identification of Klf9-regulated genes in HT22 cells by RNA-sequencing. a Treatment of HT22 [TR/TO-Klf9] cells with doxycycline (dox; 1 μg/ml) for 8 h increased Klf9 mRNA ~10 fold compared to vehicle treated cells, but had no effect in parent HT22 cells. The baseline Klf9 mRNA level did not differ between parent and [TR/TO-Klf9] line. Means with the same letter are not significantly different ( p

    Techniques Used: RNA Sequencing Assay

    64) Product Images from "Transcriptome Analysis Reveals New Insights into the Modulation of Endometrial Stromal Cell Receptive Phenotype by Embryo-Derived Signals Interleukin-1 and Human Chorionic Gonadotropin: Possible Involvement in Early Embryo Implantation"

    Article Title: Transcriptome Analysis Reveals New Insights into the Modulation of Endometrial Stromal Cell Receptive Phenotype by Embryo-Derived Signals Interleukin-1 and Human Chorionic Gonadotropin: Possible Involvement in Early Embryo Implantation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0064829

    hCG modulates IL1B effects on the expression of IL1 family members in ESCs. Confluent ESC cultures were incubated with minimal medium (MM) or hCG (100 ng/mL) for 24 h before being exposed or not to IL1B (0.1 ng/mL) for additional 24 h. Total RNA was extracted and reverse transcribed, and mRNA levels were then quantified by qRT-PCR. IL1A (A), IL1B (D), IL1RL1 (B), IL18R1 (E), IL33 (C) and IL18 (F) mRNA ratio was then determined following normalization to GAPDH mRNA (internal control). Data were from ESC cultures issued from 7 different subjects and expressed as fold change (FC) over control (ratio of IL1A, IL1B, IL1RL1, IL18R1, IL33 or IL18 mRNA levels found in cells incubated with IL1B, hCG or hCG/ILB to those found in cells incubated with MM for an equivalent period of time). *P
    Figure Legend Snippet: hCG modulates IL1B effects on the expression of IL1 family members in ESCs. Confluent ESC cultures were incubated with minimal medium (MM) or hCG (100 ng/mL) for 24 h before being exposed or not to IL1B (0.1 ng/mL) for additional 24 h. Total RNA was extracted and reverse transcribed, and mRNA levels were then quantified by qRT-PCR. IL1A (A), IL1B (D), IL1RL1 (B), IL18R1 (E), IL33 (C) and IL18 (F) mRNA ratio was then determined following normalization to GAPDH mRNA (internal control). Data were from ESC cultures issued from 7 different subjects and expressed as fold change (FC) over control (ratio of IL1A, IL1B, IL1RL1, IL18R1, IL33 or IL18 mRNA levels found in cells incubated with IL1B, hCG or hCG/ILB to those found in cells incubated with MM for an equivalent period of time). *P

    Techniques Used: Expressing, Incubation, Quantitative RT-PCR

    hCG modulates the IL1B-mediated mRNA expression of immune modulating, adhesion, growth, angiogenic and tissue remodeling factors in ESCs. Confluent ESC cultures were incubated with minimal medium (MM) (control) or hCG (100 ng/mL) for 24 h before being exposed or not to IL1B (0.1 ng/mL) for additional 24 h. Total RNA was extracted and reverse transcribed, and mRNA levels were then quantified by qRT-PCR. VCAM1 (A), IL6 (B), CCL5 (C), PTGS2 (D), VEGFC (E), MMP9 (F), TIMP3 (G) and KRT19 (H) mRNA ratio was then determined following normalization to GAPDH mRNA (internal control). Data were from ESC cultures issued from 7 different subjects and expressed as fold change (FC) over control (ratio of VCAM1, IL6, CCL5, PTGS2, VEGFC, MMP9, TIMP3 or KRT19 mRNA levels found in cells incubated with IL1B, hCG or hCG/ILB to those found in cells incubated with MM for an equivalent period of time). *P
    Figure Legend Snippet: hCG modulates the IL1B-mediated mRNA expression of immune modulating, adhesion, growth, angiogenic and tissue remodeling factors in ESCs. Confluent ESC cultures were incubated with minimal medium (MM) (control) or hCG (100 ng/mL) for 24 h before being exposed or not to IL1B (0.1 ng/mL) for additional 24 h. Total RNA was extracted and reverse transcribed, and mRNA levels were then quantified by qRT-PCR. VCAM1 (A), IL6 (B), CCL5 (C), PTGS2 (D), VEGFC (E), MMP9 (F), TIMP3 (G) and KRT19 (H) mRNA ratio was then determined following normalization to GAPDH mRNA (internal control). Data were from ESC cultures issued from 7 different subjects and expressed as fold change (FC) over control (ratio of VCAM1, IL6, CCL5, PTGS2, VEGFC, MMP9, TIMP3 or KRT19 mRNA levels found in cells incubated with IL1B, hCG or hCG/ILB to those found in cells incubated with MM for an equivalent period of time). *P

    Techniques Used: Expressing, Incubation, Quantitative RT-PCR

    hCG modulates MCPs' mRNA expression in ESCs. Confluent ESC cultures were incubated with minimal medium (MM) (control) or hCG (100 ng/mL) for 24 h before being exposed or not to IL1B (0.1 ng/mL) for additional 24 h. Total RNA was extracted and reverse transcribed. CCL2, CCL8, CCL7 and GAPDH (internal control) mRNA levels were quantified by real-time PCR. CCL2 (A), CCL8 (B) and CCL7 (C) mRNA ratio was then determined following normalization to GAPDH mRNA. Data were from ESC cultures issued from 7 different subjects and expressed as fold change (FC) over control (ratio of CCL2, CCL8 or CCL7 mRNA levels found in cells incubated with IL1B, hCG or hCG/ILB to those found in cells incubated with MM for an equivalent period of time). *P
    Figure Legend Snippet: hCG modulates MCPs' mRNA expression in ESCs. Confluent ESC cultures were incubated with minimal medium (MM) (control) or hCG (100 ng/mL) for 24 h before being exposed or not to IL1B (0.1 ng/mL) for additional 24 h. Total RNA was extracted and reverse transcribed. CCL2, CCL8, CCL7 and GAPDH (internal control) mRNA levels were quantified by real-time PCR. CCL2 (A), CCL8 (B) and CCL7 (C) mRNA ratio was then determined following normalization to GAPDH mRNA. Data were from ESC cultures issued from 7 different subjects and expressed as fold change (FC) over control (ratio of CCL2, CCL8 or CCL7 mRNA levels found in cells incubated with IL1B, hCG or hCG/ILB to those found in cells incubated with MM for an equivalent period of time). *P

    Techniques Used: Expressing, Incubation, Real-time Polymerase Chain Reaction

    65) Product Images from "Identification and Migration of Primordial Germ Cells in Atlantic Salmon, Salmo salar: Characterization of Vasa, Dead End, and Lymphocyte Antigen 75 Genes"

    Article Title: Identification and Migration of Primordial Germ Cells in Atlantic Salmon, Salmo salar: Characterization of Vasa, Dead End, and Lymphocyte Antigen 75 Genes

    Journal: Molecular Reproduction and Development

    doi: 10.1002/mrd.22142

    Distributionof vasa , dnd , and ly75 transcripts in Atlantic salmon. A : cDNA from various tissues of adult fish (blood, brain, gill, skeletal muscle, heart, liver, spleen, gall bladder, stomach, pyloric caeca, mid gut, head kidney, kidney, skin, testis, and ovary) were used for semi-quantitative RT-PCR. Actb was used as endogenous reference. Amplicon sizes, in base pairs, are indicated on the right. Expression pattern was determined using two biological replicates. B : Total RNA (400–900 ng) from early embryonic stages (two-cell, eight-cell, early-blastula, late-blastula, mid-gastrula, and 10-somite) was electrophoresed. Both 28S and 18S rRNA, stained with SYBR Safe DNA gel stain, are shown in all stages. C : The changes of both total RNA (white squares) and mRNA (black bars) amount per egg for each developmental stage. The concentration was quantified using three replicates. D : cDNA synthesized from above-mentioned developmental stages were used for semi-quantitative RT-PCR. In order to eliminate a possibility of genomic DNA contamination, −RT (without reverse transcriptase) samples of each counterpart were examined and electrophoresed. Amplicon sizes, in base pairs are indicated on the right.
    Figure Legend Snippet: Distributionof vasa , dnd , and ly75 transcripts in Atlantic salmon. A : cDNA from various tissues of adult fish (blood, brain, gill, skeletal muscle, heart, liver, spleen, gall bladder, stomach, pyloric caeca, mid gut, head kidney, kidney, skin, testis, and ovary) were used for semi-quantitative RT-PCR. Actb was used as endogenous reference. Amplicon sizes, in base pairs, are indicated on the right. Expression pattern was determined using two biological replicates. B : Total RNA (400–900 ng) from early embryonic stages (two-cell, eight-cell, early-blastula, late-blastula, mid-gastrula, and 10-somite) was electrophoresed. Both 28S and 18S rRNA, stained with SYBR Safe DNA gel stain, are shown in all stages. C : The changes of both total RNA (white squares) and mRNA (black bars) amount per egg for each developmental stage. The concentration was quantified using three replicates. D : cDNA synthesized from above-mentioned developmental stages were used for semi-quantitative RT-PCR. In order to eliminate a possibility of genomic DNA contamination, −RT (without reverse transcriptase) samples of each counterpart were examined and electrophoresed. Amplicon sizes, in base pairs are indicated on the right.

    Techniques Used: Fluorescence In Situ Hybridization, Quantitative RT-PCR, Amplification, Expressing, Staining, Concentration Assay, Synthesized

    66) Product Images from "Functional characterization of bitter-taste receptors expressed in mammalian testis"

    Article Title: Functional characterization of bitter-taste receptors expressed in mammalian testis

    Journal: Molecular Human Reproduction

    doi: 10.1093/molehr/gas040

    Tas2r gene expression in testis. ( A ) Reverse-transcription PCR covering nearly full coding sequences was performed for 9 Tas2r genes with cDNA templates reverse transcribed from mouse testis poly(A) + RNA in the presence (+) or absence (−) of reverse
    Figure Legend Snippet: Tas2r gene expression in testis. ( A ) Reverse-transcription PCR covering nearly full coding sequences was performed for 9 Tas2r genes with cDNA templates reverse transcribed from mouse testis poly(A) + RNA in the presence (+) or absence (−) of reverse

    Techniques Used: Expressing, Polymerase Chain Reaction

    67) Product Images from "The NAD(P)H Oxidase Homolog Nox4 Modulates Insulin-Stimulated Generation of H2O2 and Plays an Integral Role in Insulin Signal Transduction"

    Article Title: The NAD(P)H Oxidase Homolog Nox4 Modulates Insulin-Stimulated Generation of H2O2 and Plays an Integral Role in Insulin Signal Transduction

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.24.5.1844-1854.2004

    ). N1 to N5, Nox1 to Nox5; D1 and D2, Duox 1 and 2; G, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control; M, molecular size markers. (B) RT-PCR was performed using RNA isolated from differentiated 3T3-L1 adipocytes as described in Materials and Methods using primers for PCR analysis that were specific to murine Nox family homologs Nox1, Nox2, and Nox4. Control cell lines included CMT-93 (murine colon carcinoma cells), HL-60 (human leukemia cells), and MMC (murine mesangial cells). (C) Northern blot analysis of Nox4 mRNA expression in differentiated 3T3-L1 adipocytes. Total RNA was isolated from the indicated cells using the TRIzol reagent, and Northern blot analysis using Nox1-, Nox2-, and Nox4-specific cDNA probes or GAPDH as a loading and transfer control was performed as described in Materials and Methods.
    Figure Legend Snippet: ). N1 to N5, Nox1 to Nox5; D1 and D2, Duox 1 and 2; G, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control; M, molecular size markers. (B) RT-PCR was performed using RNA isolated from differentiated 3T3-L1 adipocytes as described in Materials and Methods using primers for PCR analysis that were specific to murine Nox family homologs Nox1, Nox2, and Nox4. Control cell lines included CMT-93 (murine colon carcinoma cells), HL-60 (human leukemia cells), and MMC (murine mesangial cells). (C) Northern blot analysis of Nox4 mRNA expression in differentiated 3T3-L1 adipocytes. Total RNA was isolated from the indicated cells using the TRIzol reagent, and Northern blot analysis using Nox1-, Nox2-, and Nox4-specific cDNA probes or GAPDH as a loading and transfer control was performed as described in Materials and Methods.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Isolation, Polymerase Chain Reaction, Northern Blot, Expressing

    ). After 48 h of incubation, cell lysates were prepared and incubated with Nox4 polyclonal antibody to immunoprecipitate Nox4 protein. Immunoprecipitates were separated by SDS-PAGE and reblotted with Nox4 antibody as described in Materials and Methods. A representative blot demonstrating expression of Nox4 protein at ∼65 kDa and a reduction in Nox4 abundance following loading of specific siRNA oligonucleotides is shown. Protein expression for the insulin receptor β-subunit is shown as an experimental control. The bar graph shows mean data from replicate immunoblots quantitated using ImageStation 440 (Kodak). *, P = 0.05; **, P = 0.01 versus control cells transfected with the scrambled siRNA. (B) At 72 h following transduction of differentiated 3T3-L1 adipocytes with adenovirus encoding β-galactosidase or the indicated Nox4 constructs, total RNA was prepared using the TRIzol reagent and RT-PCR was performed with Nox4-specific primers as described in Materials and Methods. Nox4 was detected as a ∼550-bp band in the ethidium bromide-stained agarose gel. (C) Cell lysates were prepared from 3T3-L1 cells 72 h after transduction with control adenovirus encoding β-galactosidase, wild-type PTP1B, or a site-directed catalytically inactive (Cys 215 S→er) PTP1B construct (mPTP1B). Proteins were separated by SDS-PAGE and immunoblotted with PTP1B antibody as described in Materials and Methods. The 50-kDa PTP1B protein band is indicated. Protein expression for the insulin receptor β-subunit is shown as an experimental control.
    Figure Legend Snippet: ). After 48 h of incubation, cell lysates were prepared and incubated with Nox4 polyclonal antibody to immunoprecipitate Nox4 protein. Immunoprecipitates were separated by SDS-PAGE and reblotted with Nox4 antibody as described in Materials and Methods. A representative blot demonstrating expression of Nox4 protein at ∼65 kDa and a reduction in Nox4 abundance following loading of specific siRNA oligonucleotides is shown. Protein expression for the insulin receptor β-subunit is shown as an experimental control. The bar graph shows mean data from replicate immunoblots quantitated using ImageStation 440 (Kodak). *, P = 0.05; **, P = 0.01 versus control cells transfected with the scrambled siRNA. (B) At 72 h following transduction of differentiated 3T3-L1 adipocytes with adenovirus encoding β-galactosidase or the indicated Nox4 constructs, total RNA was prepared using the TRIzol reagent and RT-PCR was performed with Nox4-specific primers as described in Materials and Methods. Nox4 was detected as a ∼550-bp band in the ethidium bromide-stained agarose gel. (C) Cell lysates were prepared from 3T3-L1 cells 72 h after transduction with control adenovirus encoding β-galactosidase, wild-type PTP1B, or a site-directed catalytically inactive (Cys 215 S→er) PTP1B construct (mPTP1B). Proteins were separated by SDS-PAGE and immunoblotted with PTP1B antibody as described in Materials and Methods. The 50-kDa PTP1B protein band is indicated. Protein expression for the insulin receptor β-subunit is shown as an experimental control.

    Techniques Used: Incubation, SDS Page, Expressing, Western Blot, Transfection, Transduction, Construct, Reverse Transcription Polymerase Chain Reaction, Staining, Agarose Gel Electrophoresis

    68) Product Images from "A conserved binding site within the Tomato Golden Mosaic Virus AL-1629 promoter is necessary for expression of viral genes important for pathogenesis"

    Article Title: A conserved binding site within the Tomato Golden Mosaic Virus AL-1629 promoter is necessary for expression of viral genes important for pathogenesis

    Journal: Virology

    doi: 10.1016/j.virol.2007.04.018

    Identification of TGMV RNA in protoplasts transfected with wild type and mutant DNA templates. The panel illustrates phosphorimager analysis of RT-PCR products after 24 cycles of amplification. Aliquots of cDNA were fractionated on agarose gels and hybridized to a probe specific for EF1α or TGMV RNA. Lanes contain RNA isolated from protoplasts transfected with AL3 [-790]-GUS template DNA containing wild type (WT), M2, M4, M5 or M6 mutant sequences. The values given below the panels represent the relative level of TGMV RNA in three experiments as determined by phosphorimager following normalization to EF1α. TGMV RNA level in wild type samples was arbitrarily assigned a value of 100%. Experiments 1 and 2 were RT-PCR reactions using two independently isolated RNA samples. Experiments 2 and 3 were independent RT-PCR reactions using the same RNA samples.
    Figure Legend Snippet: Identification of TGMV RNA in protoplasts transfected with wild type and mutant DNA templates. The panel illustrates phosphorimager analysis of RT-PCR products after 24 cycles of amplification. Aliquots of cDNA were fractionated on agarose gels and hybridized to a probe specific for EF1α or TGMV RNA. Lanes contain RNA isolated from protoplasts transfected with AL3 [-790]-GUS template DNA containing wild type (WT), M2, M4, M5 or M6 mutant sequences. The values given below the panels represent the relative level of TGMV RNA in three experiments as determined by phosphorimager following normalization to EF1α. TGMV RNA level in wild type samples was arbitrarily assigned a value of 100%. Experiments 1 and 2 were RT-PCR reactions using two independently isolated RNA samples. Experiments 2 and 3 were independent RT-PCR reactions using the same RNA samples.

    Techniques Used: Transfection, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Amplification, Isolation

    69) Product Images from "Oxidative stress-responsive microRNA-320 regulates glycolysis in diverse biological systems"

    Article Title: Oxidative stress-responsive microRNA-320 regulates glycolysis in diverse biological systems

    Journal: The FASEB Journal

    doi: 10.1096/fj.11-197467

    Mir-320a regulates glycolysis in vitro and in vivo. A ) Reduction of miR-320a increases lactate level. Anti-miR-320a was transfected into C2C12 cells, and the intracellular level of lactate was measured ( n =3). B ) Lack of mir-320a sensitizes cells to oxidative stress-induced lactate production. Anti-miR-320a was transfected into C2C12 cells; 3 d later, the cells were treated with H 2 O 2 40 μM for 24 h. Lactate level was measured. Note that anti-miR-320a-transfected cells accumulate more lactate in response to H 2 O 2 treatment ( n =3). C , D ) Reduction in miR-320a is required for oxidative stress-induced glycolysis and the expression of PFKm. Transfected C2C12 cells (either control or pre-miR-320a, 40 nM) were treated with H 2 O 2 (40 μm, 24 h). Lactate level was measured ( C ) and PFKm was detected by Western blot and quantitative data was acquired by normalizing to actin ( D ). E–G ) MiR-320a regulates glycolysis in vivo . MiR-320a (320 nmol) was electroporated into TA muscle, together with GFP as reporter. E ) Whole TA muscle under fluorescent microscope; white portion is the region of GFP + fibers. GFP + muscle fibers were isolated for both RNA and protein preparation. Quantitative PCR was used to detect the level of miR-320a in skeletal muscle ( E ), and equal amounts of total proteins were subjected to biochemical assay to detect the level of lactate ( F ) and Western blot analysis for detection of PFKm ( G ). Both actin and total protein (Ponceau S staining) are shown as controls. * P
    Figure Legend Snippet: Mir-320a regulates glycolysis in vitro and in vivo. A ) Reduction of miR-320a increases lactate level. Anti-miR-320a was transfected into C2C12 cells, and the intracellular level of lactate was measured ( n =3). B ) Lack of mir-320a sensitizes cells to oxidative stress-induced lactate production. Anti-miR-320a was transfected into C2C12 cells; 3 d later, the cells were treated with H 2 O 2 40 μM for 24 h. Lactate level was measured. Note that anti-miR-320a-transfected cells accumulate more lactate in response to H 2 O 2 treatment ( n =3). C , D ) Reduction in miR-320a is required for oxidative stress-induced glycolysis and the expression of PFKm. Transfected C2C12 cells (either control or pre-miR-320a, 40 nM) were treated with H 2 O 2 (40 μm, 24 h). Lactate level was measured ( C ) and PFKm was detected by Western blot and quantitative data was acquired by normalizing to actin ( D ). E–G ) MiR-320a regulates glycolysis in vivo . MiR-320a (320 nmol) was electroporated into TA muscle, together with GFP as reporter. E ) Whole TA muscle under fluorescent microscope; white portion is the region of GFP + fibers. GFP + muscle fibers were isolated for both RNA and protein preparation. Quantitative PCR was used to detect the level of miR-320a in skeletal muscle ( E ), and equal amounts of total proteins were subjected to biochemical assay to detect the level of lactate ( F ) and Western blot analysis for detection of PFKm ( G ). Both actin and total protein (Ponceau S staining) are shown as controls. * P

    Techniques Used: In Vitro, In Vivo, Transfection, Expressing, Western Blot, Microscopy, Isolation, Real-time Polymerase Chain Reaction, Staining

    70) Product Images from "The m6A Reader ECT2 Controls Trichome Morphology by Affecting mRNA Stability in Arabidopsis [OPEN]"

    Article Title: The m6A Reader ECT2 Controls Trichome Morphology by Affecting mRNA Stability in Arabidopsis [OPEN]

    Journal: The Plant Cell

    doi: 10.1105/tpc.17.00934

    ECT2 Recognizes URUAY, a Plant-Specific 3′ UTR m 6 A Motif That Can Be Methylated by Arabidopsis Endogenous m 6 A Writer Proteins. (A) TLC results from an in vitro methylation assay using nuclear extracts with site specific labeled substrates. pA indicates [γ- 32 P]-labeled adenosine. Oligo 1, 5′-CUCGAUCCUUUUUGUpAGUUUCCGAC-3′; Oligo 2, 5′-UAUGCGUCUACUGUpACGGUUGAAUUU-3′. (B) TLC results from an in vitro methylation assay using nuclear extracts with site-specific labeled oligo RNA substrates. The RNA probes were as follows, and pA indicates [γ- 32 P]-labeled adenosine. UGUpA, 5′-CUCGAUCCUUUUUGUpAGUUUCCGAC-3′; GGpACU, 5′-CUCGAUCCUUUUGGpACUGUUUCCGAC-3′; CUpAUG, 5′-CUCGAUCCUUUUCUpAUGGUUUCCGAC-3′. (C) Quantification of m 6 A/A ratios in (B) , as calculated by densitometry using Image J. (D) EMSA measuring the dissociation constant ( K d , nM) of GST-ECT2 with methylated and unmethylated RNA probes. The 4 nmol RNA probe was labeled with [γ- 32 P], and GST-ECT2 concentration ranged from 10 to 2000 nM. Oligo RNA, 5′-AUGGGCCGUUCAUCUGCUAAAA(GGXCU/UGUXA/CUXUG) GCUUUUGGGGCUU*G*U-3′, X = A/m 6 A. The asterisk indicates that thiol-protected bases were used for the experiment.
    Figure Legend Snippet: ECT2 Recognizes URUAY, a Plant-Specific 3′ UTR m 6 A Motif That Can Be Methylated by Arabidopsis Endogenous m 6 A Writer Proteins. (A) TLC results from an in vitro methylation assay using nuclear extracts with site specific labeled substrates. pA indicates [γ- 32 P]-labeled adenosine. Oligo 1, 5′-CUCGAUCCUUUUUGUpAGUUUCCGAC-3′; Oligo 2, 5′-UAUGCGUCUACUGUpACGGUUGAAUUU-3′. (B) TLC results from an in vitro methylation assay using nuclear extracts with site-specific labeled oligo RNA substrates. The RNA probes were as follows, and pA indicates [γ- 32 P]-labeled adenosine. UGUpA, 5′-CUCGAUCCUUUUUGUpAGUUUCCGAC-3′; GGpACU, 5′-CUCGAUCCUUUUGGpACUGUUUCCGAC-3′; CUpAUG, 5′-CUCGAUCCUUUUCUpAUGGUUUCCGAC-3′. (C) Quantification of m 6 A/A ratios in (B) , as calculated by densitometry using Image J. (D) EMSA measuring the dissociation constant ( K d , nM) of GST-ECT2 with methylated and unmethylated RNA probes. The 4 nmol RNA probe was labeled with [γ- 32 P], and GST-ECT2 concentration ranged from 10 to 2000 nM. Oligo RNA, 5′-AUGGGCCGUUCAUCUGCUAAAA(GGXCU/UGUXA/CUXUG) GCUUUUGGGGCUU*G*U-3′, X = A/m 6 A. The asterisk indicates that thiol-protected bases were used for the experiment.

    Techniques Used: Methylation, Thin Layer Chromatography, In Vitro, Labeling, Concentration Assay

    Transcriptome-Wide ECT2-RNA Interaction Sites Reveal a Unique Binding Motif and 3′ UTR Enrichment. (A) Schematic diagram of the FA-CLIP method. FA-CLIP procedure: A cell extract from formaldehyde-cross-linked 14-d-old ECT2 pro :ECT2-Flag/ect2-1 seedlings was subjected to immunoprecipitation using anti-Flag M2 magnetic beads. Two on-bead digestions using RNase T1 and Protease K were performed sequentially: ECT2 protein-bound transcripts were digested into protein binding regions (∼30 nucleotides) using RNase T1, and ECT2 protein was digested by Protease K to leave an amino acid tag (that is, cross-linked residue) on ECT2-bound RNA fragments, which will induce mutation by reverse transcription. ECT2-bound RNA fragments were isolated, converted to cDNA (harboring mutations), and sequenced (termed FA-CLIP-ECT2). FA-CLIP of wild-type Col-0 (termed Mock) was also performed as described for FA-CLIP-ECT2. Total RNA with rRNA depletion from ECT2 pro : ECT2-Flag/ect2-1 seedlings was used for RNA-seq (termed Input). IP enrichment-based ECT2 binding peaks (termed FA-CLIP enrichment peaks) were obtained by subtracting the enrichment peaks in Mock (Mock versus Input) from the enrichment peaks in FA-CLIP-ECT2 (FA-CLIP-ECT2 versus Input). The mutation-based ECT2 binding peaks (termed FA-CLIP mutation peaks) are determined by subtracting the mutation peaks in Mock from the mutation peaks in FA-CLIP-ECT2. ECT2 binding peaks (termed “FA-CLIP peaks”) are defined as overlapping FA-CLIP mutation peaks and FA-CLIP enrichment peaks. (B) Overlap of two biological replicates of the number of FA-CLIP peaks identifying ∼6000 high-confidence ECT2 binding sites (i.e., ECT2 targets) corresponding to 3680 unique transcripts/genes. Biological replicates are parallel measurements of biologically distinct samples. (C) Pie chart presenting RNA types (that is, transcript species) of ECT2-bound transcripts. (D) Overlap of the identified ECT2 binding peaks (FA-CLIP peaks) and m 6 A peaks. (E) Pie chart presenting the fraction of ECT2 binding peaks in each of the three non-overlapping transcript segments (5′ UTR, coding sequence [CDS], and 3′ UTR). (F) Metagene profiles of the distribution of ECT2 binding peaks along a normalized transcript composed of three rescaled nonoverlapping segments listed on the x axis. (G) Binding motif identified by HOMER based on all identified ECT2 binding peaks (6047 peaks; P = 1 × 10 −215 ).
    Figure Legend Snippet: Transcriptome-Wide ECT2-RNA Interaction Sites Reveal a Unique Binding Motif and 3′ UTR Enrichment. (A) Schematic diagram of the FA-CLIP method. FA-CLIP procedure: A cell extract from formaldehyde-cross-linked 14-d-old ECT2 pro :ECT2-Flag/ect2-1 seedlings was subjected to immunoprecipitation using anti-Flag M2 magnetic beads. Two on-bead digestions using RNase T1 and Protease K were performed sequentially: ECT2 protein-bound transcripts were digested into protein binding regions (∼30 nucleotides) using RNase T1, and ECT2 protein was digested by Protease K to leave an amino acid tag (that is, cross-linked residue) on ECT2-bound RNA fragments, which will induce mutation by reverse transcription. ECT2-bound RNA fragments were isolated, converted to cDNA (harboring mutations), and sequenced (termed FA-CLIP-ECT2). FA-CLIP of wild-type Col-0 (termed Mock) was also performed as described for FA-CLIP-ECT2. Total RNA with rRNA depletion from ECT2 pro : ECT2-Flag/ect2-1 seedlings was used for RNA-seq (termed Input). IP enrichment-based ECT2 binding peaks (termed FA-CLIP enrichment peaks) were obtained by subtracting the enrichment peaks in Mock (Mock versus Input) from the enrichment peaks in FA-CLIP-ECT2 (FA-CLIP-ECT2 versus Input). The mutation-based ECT2 binding peaks (termed FA-CLIP mutation peaks) are determined by subtracting the mutation peaks in Mock from the mutation peaks in FA-CLIP-ECT2. ECT2 binding peaks (termed “FA-CLIP peaks”) are defined as overlapping FA-CLIP mutation peaks and FA-CLIP enrichment peaks. (B) Overlap of two biological replicates of the number of FA-CLIP peaks identifying ∼6000 high-confidence ECT2 binding sites (i.e., ECT2 targets) corresponding to 3680 unique transcripts/genes. Biological replicates are parallel measurements of biologically distinct samples. (C) Pie chart presenting RNA types (that is, transcript species) of ECT2-bound transcripts. (D) Overlap of the identified ECT2 binding peaks (FA-CLIP peaks) and m 6 A peaks. (E) Pie chart presenting the fraction of ECT2 binding peaks in each of the three non-overlapping transcript segments (5′ UTR, coding sequence [CDS], and 3′ UTR). (F) Metagene profiles of the distribution of ECT2 binding peaks along a normalized transcript composed of three rescaled nonoverlapping segments listed on the x axis. (G) Binding motif identified by HOMER based on all identified ECT2 binding peaks (6047 peaks; P = 1 × 10 −215 ).

    Techniques Used: Binding Assay, Cross-linking Immunoprecipitation, Immunoprecipitation, Magnetic Beads, Protein Binding, Mutagenesis, Isolation, RNA Sequencing Assay, Sequencing

    71) Product Images from "Taxonomic Assessment of Rumen Microbiota Using Total RNA and Targeted Amplicon Sequencing Approaches"

    Article Title: Taxonomic Assessment of Rumen Microbiota Using Total RNA and Targeted Amplicon Sequencing Approaches

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2016.00987

    Microbial community composition estimated in the RNA-seq and RNA/DNA Amplicon-seq datasets . Microbial community composition of (A) bacterial phyla, (B) bacterial families, (C) archaea, and (D) dataset-specific taxa.
    Figure Legend Snippet: Microbial community composition estimated in the RNA-seq and RNA/DNA Amplicon-seq datasets . Microbial community composition of (A) bacterial phyla, (B) bacterial families, (C) archaea, and (D) dataset-specific taxa.

    Techniques Used: RNA Sequencing Assay, Amplification

    Flow chart of the pipeline for analyzing rumen microbiota using RNA-seq and RNA/DNA Amplicon-seq . The regionally enriched Greengenes 16S rRNA gene database (version gg_13_5_99 accessed May 2013) was used to analyze the bacterial community, and the regionally enriched rumen-specific archaeal database was used to analyze the archaeal community.
    Figure Legend Snippet: Flow chart of the pipeline for analyzing rumen microbiota using RNA-seq and RNA/DNA Amplicon-seq . The regionally enriched Greengenes 16S rRNA gene database (version gg_13_5_99 accessed May 2013) was used to analyze the bacterial community, and the regionally enriched rumen-specific archaeal database was used to analyze the archaeal community.

    Techniques Used: Flow Cytometry, RNA Sequencing Assay, Amplification

    Dissimilarities among the RNA-seq, RNA Amplicon-seq and DNA Amplicon-seq datasets revealed by principal coordinate analysis (PCoA). (A) PCoA based on shared bacterial phyla, (B) PCoA based on shared bacterial families, (C) PCoA based on shared archaeal mixed taxa. PCoA was performed based on the Bray-Curtis dissimilarity matrix.
    Figure Legend Snippet: Dissimilarities among the RNA-seq, RNA Amplicon-seq and DNA Amplicon-seq datasets revealed by principal coordinate analysis (PCoA). (A) PCoA based on shared bacterial phyla, (B) PCoA based on shared bacterial families, (C) PCoA based on shared archaeal mixed taxa. PCoA was performed based on the Bray-Curtis dissimilarity matrix.

    Techniques Used: RNA Sequencing Assay, Amplification

    Co-occurrence of abundant microbial taxa in the RNA-seq and DNA Amplicon-seq datasets. (A) Correlation matrix of abundant microbial taxa and (B) Network of abundant microbial taxa in the RNA-seq dataset. Only bacterial families and archeael taxa with a relative abundance > 0.1% and detected in all five rumen samples using both RNA-seq and DNA Amplicon-seq were analyzed using Spearman's rank correlation. The RNA Amplicon-seq dataset was not included into the analysis, because its bacterial and archaeal profiles were similar to profiles from the RNA-seq dataset. In (A) , the sub-matrix surrounded by the black square exhibits correlations between taxa in the RNA-seq and DNA Amplicon-seq datasets. Strong correlations (Spearman's rank correlation coefficient [ρ] ≥ 0.9 or ≤ −0.9) were displayed with * (0.05
    Figure Legend Snippet: Co-occurrence of abundant microbial taxa in the RNA-seq and DNA Amplicon-seq datasets. (A) Correlation matrix of abundant microbial taxa and (B) Network of abundant microbial taxa in the RNA-seq dataset. Only bacterial families and archeael taxa with a relative abundance > 0.1% and detected in all five rumen samples using both RNA-seq and DNA Amplicon-seq were analyzed using Spearman's rank correlation. The RNA Amplicon-seq dataset was not included into the analysis, because its bacterial and archaeal profiles were similar to profiles from the RNA-seq dataset. In (A) , the sub-matrix surrounded by the black square exhibits correlations between taxa in the RNA-seq and DNA Amplicon-seq datasets. Strong correlations (Spearman's rank correlation coefficient [ρ] ≥ 0.9 or ≤ −0.9) were displayed with * (0.05

    Techniques Used: RNA Sequencing Assay, Amplification

    72) Product Images from "Identification of crassin acetate as a new immunosuppressant triggering heme oxygenase-1 expression in dendritic cells"

    Article Title: Identification of crassin acetate as a new immunosuppressant triggering heme oxygenase-1 expression in dendritic cells

    Journal: Blood

    doi: 10.1182/blood-2009-02-204297

    CRA-induced HO-1 expression in DCs . (A) BM-DCs were treated with CRA (10 μM) in the presence of vehicle alone or LPS (10 ng/mL) for 6 hours. Total RNA was isolated from these cells, and subjected to real-time PCR analysis for mRNA expression for HO-1 and GAPDH (mean ± SD, n = 3). Data are normalized to GAPDH mRNA expression as mentioned in “Measurement of in vitro impacts of CRA on LPS-induced DC maturation.” (B-C) BM-DCs were cultured with CRA in the presence of vehicle alone or LPS to examine time kinetics (10 μM; B) and dose dependency (6 hours) for HO-1 protein expression (C). The whole-cell extracts were assessed by Western blotting for HO-1 protein expression. The whole-cell extracts were also subjected to Coomassie blue staining. (D) HO-1 enzymatic activity was examined in whole-cell extracts from BM-DCs treated with CRA or vehicle alone in the presence or absence of LPS for 6 hours. Data are shown as nmol of bilirubin/minute per mg protein (mean ± SD, n = 3). (E-F) CD3 + T cells freshly purified from C57BL/6 mice were treated with CRA (10 μM) or CoPPIX (50 μM) in the presence (F) or absence (E) of PMA plus ionomycin for 6 hours and then examined for HO-1 mRNA expression by real-time PCR. Data are normalized to GAPDH mRNA expression (mean ± SD, n = 3). Asterisks indicate statistically significant (** P
    Figure Legend Snippet: CRA-induced HO-1 expression in DCs . (A) BM-DCs were treated with CRA (10 μM) in the presence of vehicle alone or LPS (10 ng/mL) for 6 hours. Total RNA was isolated from these cells, and subjected to real-time PCR analysis for mRNA expression for HO-1 and GAPDH (mean ± SD, n = 3). Data are normalized to GAPDH mRNA expression as mentioned in “Measurement of in vitro impacts of CRA on LPS-induced DC maturation.” (B-C) BM-DCs were cultured with CRA in the presence of vehicle alone or LPS to examine time kinetics (10 μM; B) and dose dependency (6 hours) for HO-1 protein expression (C). The whole-cell extracts were assessed by Western blotting for HO-1 protein expression. The whole-cell extracts were also subjected to Coomassie blue staining. (D) HO-1 enzymatic activity was examined in whole-cell extracts from BM-DCs treated with CRA or vehicle alone in the presence or absence of LPS for 6 hours. Data are shown as nmol of bilirubin/minute per mg protein (mean ± SD, n = 3). (E-F) CD3 + T cells freshly purified from C57BL/6 mice were treated with CRA (10 μM) or CoPPIX (50 μM) in the presence (F) or absence (E) of PMA plus ionomycin for 6 hours and then examined for HO-1 mRNA expression by real-time PCR. Data are normalized to GAPDH mRNA expression (mean ± SD, n = 3). Asterisks indicate statistically significant (** P

    Techniques Used: Expressing, Isolation, Real-time Polymerase Chain Reaction, In Vitro, Cell Culture, Western Blot, Staining, Activity Assay, Purification, Mouse Assay

    73) Product Images from "Human Melanoma Cells Do Not Express Fas (Apo-1/CD95) Ligand"

    Article Title: Human Melanoma Cells Do Not Express Fas (Apo-1/CD95) Ligand

    Journal: Cancer research

    doi:

    Melanoma cell lines do not express FasL mRNA by RT-PCR. Total RNA was isolated from cultured melanoma lines, the antimelanoma T-cell line 1143, Jurkat cells, and the FasL + colon carcinoma SW480 cells (TRIzol; Life Technologies, Inc.). The RT reaction was followed by 40 cycles of PCR using intron-spanning primers. β -Actin was amplified as a positive control for each cell line. Lanes +, addition of SuperScript II to the RT reaction; Lanes −, absence of SuperScript II in the RT reaction. A, a 344-bp fragment corresponding to FasL mRNA was observed in the antimelanoma T cell line 1143 after growing in IL-2 and after TCR cross-linking with OKT3. FasL mRNA was observed in OKT3 activated Jurkat cells but not resting Jurkat cells. Additionally, the 344-bp fragment representing FasL mRNA was detected in the colon carcinoma SW480 but not in any of the 26 melanoma cell lines tested in four separate experiments. B , a nonoverlapping panel of melanoma lines demonstrating the absence of the 344-bp fragment that corresponds to FasL.
    Figure Legend Snippet: Melanoma cell lines do not express FasL mRNA by RT-PCR. Total RNA was isolated from cultured melanoma lines, the antimelanoma T-cell line 1143, Jurkat cells, and the FasL + colon carcinoma SW480 cells (TRIzol; Life Technologies, Inc.). The RT reaction was followed by 40 cycles of PCR using intron-spanning primers. β -Actin was amplified as a positive control for each cell line. Lanes +, addition of SuperScript II to the RT reaction; Lanes −, absence of SuperScript II in the RT reaction. A, a 344-bp fragment corresponding to FasL mRNA was observed in the antimelanoma T cell line 1143 after growing in IL-2 and after TCR cross-linking with OKT3. FasL mRNA was observed in OKT3 activated Jurkat cells but not resting Jurkat cells. Additionally, the 344-bp fragment representing FasL mRNA was detected in the colon carcinoma SW480 but not in any of the 26 melanoma cell lines tested in four separate experiments. B , a nonoverlapping panel of melanoma lines demonstrating the absence of the 344-bp fragment that corresponds to FasL.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Isolation, Cell Culture, Polymerase Chain Reaction, Amplification, Positive Control

    74) Product Images from "Oncogenic homeodomain transcription factor E2A-Pbx1 activates a novel WNT gene in pre-B acute lymphoblastoid leukemia"

    Article Title: Oncogenic homeodomain transcription factor E2A-Pbx1 activates a novel WNT gene in pre-B acute lymphoblastoid leukemia

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    Treatment of t(1;19)-positive pre-B acute lymphoblastoid cell lines with antisense 2′-methoxyethyl phosphorothioate oligonucleotides to E2A-Pbx1 reveals that WNT-16 transcript levels are dependent on the presence of E2A-Pbx1. 697 cells were electroporated with antisense oligonucleotides with specificity for E2A-Pbx1. Twenty hours later, total RNA was derived from the cells and analyzed by Northern blotting by using asymmetric PCR probes for E2A-Pbx1 and WNT-16 and a random primer-labeled cDNA probe for GAPDH. ( A ) Northern blots. Nalm-6 (N-6) RNA was included as a control. ( B ) Graphical representation of Northern blot results. Relative mRNA levels detected on Northern blots were quantitated by using a PhosphorImager. Bar = mean ± SD from three independent experiments.
    Figure Legend Snippet: Treatment of t(1;19)-positive pre-B acute lymphoblastoid cell lines with antisense 2′-methoxyethyl phosphorothioate oligonucleotides to E2A-Pbx1 reveals that WNT-16 transcript levels are dependent on the presence of E2A-Pbx1. 697 cells were electroporated with antisense oligonucleotides with specificity for E2A-Pbx1. Twenty hours later, total RNA was derived from the cells and analyzed by Northern blotting by using asymmetric PCR probes for E2A-Pbx1 and WNT-16 and a random primer-labeled cDNA probe for GAPDH. ( A ) Northern blots. Nalm-6 (N-6) RNA was included as a control. ( B ) Graphical representation of Northern blot results. Relative mRNA levels detected on Northern blots were quantitated by using a PhosphorImager. Bar = mean ± SD from three independent experiments.

    Techniques Used: Derivative Assay, Northern Blot, Polymerase Chain Reaction, Labeling

    Expression of Frizzled genes in human B cell lines. RNA was derived from human pre-B cells carrying the t(1;19) translocation (697, KOPN-36, KOPN-63), pro-B cells (UOCB-1), pre-B cells (Nalm-6, Reh), mature B cells (Namalwa and Ramos), and a T lineage cell (Jurkat). Yeast tRNA is included as a control. RNA was analyzed by RNase protection by using antisense transcripts of FZ-2, FZ-3, FZ-5, and FZ-6 as probes. P, labeled probe; PF, protected fragment.
    Figure Legend Snippet: Expression of Frizzled genes in human B cell lines. RNA was derived from human pre-B cells carrying the t(1;19) translocation (697, KOPN-36, KOPN-63), pro-B cells (UOCB-1), pre-B cells (Nalm-6, Reh), mature B cells (Namalwa and Ramos), and a T lineage cell (Jurkat). Yeast tRNA is included as a control. RNA was analyzed by RNase protection by using antisense transcripts of FZ-2, FZ-3, FZ-5, and FZ-6 as probes. P, labeled probe; PF, protected fragment.

    Techniques Used: Expressing, Derivative Assay, Translocation Assay, Labeling

    75) Product Images from "Murine osteoclasts secrete serine protease HtrA1 capable of degrading osteoprotegerin in the bone microenvironment"

    Article Title: Murine osteoclasts secrete serine protease HtrA1 capable of degrading osteoprotegerin in the bone microenvironment

    Journal: Communications Biology

    doi: 10.1038/s42003-019-0334-5

    Screening of candidates of osteoprotegerin (OPG)-degrading enzymes by RNA-sequencing analysis. a RNA was extracted from bone marrow macrophages (BMMs) and osteoclasts (OCs) and subjected to RNA-sequencing analysis. Gene expression levels in BMMs and OCs were plotted on the horizontal and vertical axis, respectively. The intensity of expression is shown as log 2 -transformed RPKM (reads per kilobase of exon model per million mapped reads) values. Gray dots: genes detected by the RNA-sequencing analysis. Red dots: genes more strongly or weakly expressed in OCs than in BMMs ( > 3 SD). Black dots: genes coding for proteases. b Venn diagram showing overlap between genes identified by mass spectrometry (Table 1 ) and RNA-sequencing analysis (Table 2). c The expression of Htra1 and Mmp9 , as well as osteoclast markers ( Ctsk, Acp5 , and Oscar ) in BMMs and OCs was assessed by quantitative RT-PCR. Data are expressed as means ± SD ( n = 3). Htra1 : t 4 = −13.5, p = 1.7 × 10 −4 , 95% confidence interval (CI) [−6.2, −4.1]; Mmp9 : t 4 = −8.1, p = 0.0013, 95% CI [−8.2, −4.0]; Acp5 : t 4 = −11.6, p = 3.1 × 10 −4 , 95% CI [−4.8, −3.0]; Ctsk : t 4 = −6.6, p = 0.0028, 95% CI [−3.8, −1.6]; Oscar : t 4 = −6.5, p = 0.0029, 95% CI [−11.9, −4.8]. Data are expressed as means ± SD ( n = 3). ** p
    Figure Legend Snippet: Screening of candidates of osteoprotegerin (OPG)-degrading enzymes by RNA-sequencing analysis. a RNA was extracted from bone marrow macrophages (BMMs) and osteoclasts (OCs) and subjected to RNA-sequencing analysis. Gene expression levels in BMMs and OCs were plotted on the horizontal and vertical axis, respectively. The intensity of expression is shown as log 2 -transformed RPKM (reads per kilobase of exon model per million mapped reads) values. Gray dots: genes detected by the RNA-sequencing analysis. Red dots: genes more strongly or weakly expressed in OCs than in BMMs ( > 3 SD). Black dots: genes coding for proteases. b Venn diagram showing overlap between genes identified by mass spectrometry (Table 1 ) and RNA-sequencing analysis (Table 2). c The expression of Htra1 and Mmp9 , as well as osteoclast markers ( Ctsk, Acp5 , and Oscar ) in BMMs and OCs was assessed by quantitative RT-PCR. Data are expressed as means ± SD ( n = 3). Htra1 : t 4 = −13.5, p = 1.7 × 10 −4 , 95% confidence interval (CI) [−6.2, −4.1]; Mmp9 : t 4 = −8.1, p = 0.0013, 95% CI [−8.2, −4.0]; Acp5 : t 4 = −11.6, p = 3.1 × 10 −4 , 95% CI [−4.8, −3.0]; Ctsk : t 4 = −6.6, p = 0.0028, 95% CI [−3.8, −1.6]; Oscar : t 4 = −6.5, p = 0.0029, 95% CI [−11.9, −4.8]. Data are expressed as means ± SD ( n = 3). ** p

    Techniques Used: RNA Sequencing Assay, Expressing, Transformation Assay, Mass Spectrometry, Quantitative RT-PCR

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    Microarray:

    Article Title: Surfactant Protein D Reverses the Gene Signature of Transepithelial HIV-1 Passage and Restricts the Viral Transfer Across the Vaginal Barrier
    Article Snippet: Validation by Real Time RT-PCR Since, EpiVaginal tissues (of ectocervical origin) used in the assays were sufficient enough for microarray analysis, we carried out the validation of microarray data using an ectocervical cell line (Ect1/E6E7) under conditions similar to the ex vivo model of HIV-1 transmission (same MOI). .. Cells were seeded in a 96-well plate, grown up to confluence and then treated with rfhSP-D (100 μg/ml) for 20 min, before inoculation with 100 TCID50 R5 tropic HIV-1JR−CSF at 37°C for 24 h. Total RNA was isolated using Trizol (Invitrogen) and the quality of RNA was assessed by nano-spectrophotometry and the nucleotide: protein ratio (260:280) was determined.

    Article Title: Transcriptome Analysis Reveals New Insights into the Modulation of Endometrial Stromal Cell Receptive Phenotype by Embryo-Derived Signals Interleukin-1 and Human Chorionic Gonadotropin: Possible Involvement in Early Embryo Implantation
    Article Snippet: .. Gene expression profile for all experiments Total RNA was isolated from primary human ESCs treated with hCG (100 ng/mL) for 24 h before being stimulated or not with IL1B (0.1 ng/mL) for additional 24 h. RNA from all treated groups was compared by micro-array with the corresponding non-treated control using Affymetrix GeneChip Human Genome. ..

    Article Title: Socs3 maintains the specificity of biological responses to cytokine signals during granulocyte and macrophage differentiation
    Article Snippet: .. To validate the differential expression of genes identified by microarray analysis, two independent Lin− Kit+ hematopoietic progenitor cell samples were treated with 100 ng/mL IL-6, 10 ng/mL G-CSF or saline for 4 h, and total RNA was prepared using TRIzol and RNeasy columns. cDNA was generated using Superscript II reverse transcriptase (Invitrogen, CA, USA) according to the manufacturer's instructions. .. Quantitative real-time PCR was performed using an Applied Biosystems GeneCard and a 7900HT scanner (Applied Biosystems, CA, USA).

    Incubation:

    Article Title: TNF-alpha upregulates the A2B adenosine receptor gene: the role of NAD(P)H oxidase 4
    Article Snippet: Total RNA from aortic VSMC was prepared with Trizol® (Invitrogen, Carlsbad, Ca) according to the manufacturer’s instructions [ ]. .. When inhibitors were use in conjunction with another stimulus (Ad-β-gal, Ad-Nox4, Ad-DN-Nox4 adenoviruses, or TNF-α, inhibitors were incubated with cells for 1 hour before the stimulus was added.

    Expressing:

    Article Title: Piwi-Interacting RNA1037 Enhances Chemoresistance and Motility in Human Oral Squamous Cell Carcinoma Cells
    Article Snippet: .. For the quantitative assessment of XIAP mRNA expression, total RNA from the cells was extracted using Trizol (Invitrogen, USA). .. First-strand cDNA was synthesized from 1 μg of RNA using the RevertAid First Strand cDNA synthesis kit from Thermo Fisher Scientific, USA.

    Article Title: SRPX2 regulates colon cancer cell metabolism by miR-192/215 via PI3K-Akt
    Article Snippet: Total RNA and miRNA was extracted from the colon cancer cells using Trizol (Invitrogen, CA, USA) based on the protocol. .. For mRNA and miRNA expression detection, real time RT-PCR was performed usinga sequence detector (ABI-Prism, Applied Biosystems) following the instructions.

    Article Title: A MicroRNA196a2* and TP63 Circuit Regulated by Estrogen Receptor-? and ERK2 that Controls Breast Cancer Proliferation and Invasiveness Properties
    Article Snippet: .. For the analysis of miRNA expression, total RNA was isolated, reverse transcribed using primers specific for each miRNA (Applied Biosystems, Foster, CA) and analyzed by real-time PCR using Taqman chemistry and primers from Applied Biosystems. .. Total RNA was isolated using Trizol reagent.

    Article Title: Star-PAP regulates tumor protein D52 through modulating miR-449a/34a in breast cancer
    Article Snippet: For detection of mature miR-449a/34a, total RNA was subjected to reverse TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems), and TaqMan MicroRNA Assay Kit (Applied Biosystems) was used for qPCR analysis. .. Relative changes in gene and miRNA expression were determined using the 2−ΔΔ Ct method.

    Article Title: Transcriptome Analysis Reveals New Insights into the Modulation of Endometrial Stromal Cell Receptive Phenotype by Embryo-Derived Signals Interleukin-1 and Human Chorionic Gonadotropin: Possible Involvement in Early Embryo Implantation
    Article Snippet: .. Gene expression profile for all experiments Total RNA was isolated from primary human ESCs treated with hCG (100 ng/mL) for 24 h before being stimulated or not with IL1B (0.1 ng/mL) for additional 24 h. RNA from all treated groups was compared by micro-array with the corresponding non-treated control using Affymetrix GeneChip Human Genome. ..

    Article Title: TNF-alpha upregulates the A2B adenosine receptor gene: the role of NAD(P)H oxidase 4
    Article Snippet: Paragraph title: Measurement of A2B AR mRNA expression ... Total RNA from aortic VSMC was prepared with Trizol® (Invitrogen, Carlsbad, Ca) according to the manufacturer’s instructions [ ].

    Article Title: Socs3 maintains the specificity of biological responses to cytokine signals during granulocyte and macrophage differentiation
    Article Snippet: .. To validate the differential expression of genes identified by microarray analysis, two independent Lin− Kit+ hematopoietic progenitor cell samples were treated with 100 ng/mL IL-6, 10 ng/mL G-CSF or saline for 4 h, and total RNA was prepared using TRIzol and RNeasy columns. cDNA was generated using Superscript II reverse transcriptase (Invitrogen, CA, USA) according to the manufacturer's instructions. .. Quantitative real-time PCR was performed using an Applied Biosystems GeneCard and a 7900HT scanner (Applied Biosystems, CA, USA).

    Article Title: Adaptations to chronic rapamycin in mice
    Article Snippet: .. Respective cDNAs were then synthesized from 2 µg total RNA using ABI's TaqMan RNA-to-Ct Kit, and qRT-PCR was performed on 25 ng cDNA in a 10 µl reaction (2.5 ng for 18S only) using single tube inventoried TaqMan probes and TaqMan Gene Expression Master Mix (Life Technologies). .. The TaqMan primer sequences are B2m,mCG11606, Mm00437762_m1 and FG,18S rRNA, 4333760T. qRT-PCR was performed to detect Rpl22 and Rpl22l1 on 25 ng cDNA in a 10 µl reaction using SYBR Green (Biorad cat. 1725271).

    Article Title: Choline Induces Transcriptional Repression and Proteasomal Degradation of the Malarial Phosphoethanolamine Methyltransferase ▿
    Article Snippet: Wild-type 3D7 and transgenic parasites constitutively expressing Pf PMT under the calmodulin promoter (TP-CAM-PfPMT) were synchronized with 5% d -sorbitol and grown to early trophozoite stage in medium without choline. .. For real time reverse transcription-PCR (RT-PCR) analysis, total RNA was extracted from the parasite pellets by using the TRIzol reagent (Invitrogen) following the manufacturer's instructions.

    Ex Vivo:

    Article Title: Surfactant Protein D Reverses the Gene Signature of Transepithelial HIV-1 Passage and Restricts the Viral Transfer Across the Vaginal Barrier
    Article Snippet: Validation by Real Time RT-PCR Since, EpiVaginal tissues (of ectocervical origin) used in the assays were sufficient enough for microarray analysis, we carried out the validation of microarray data using an ectocervical cell line (Ect1/E6E7) under conditions similar to the ex vivo model of HIV-1 transmission (same MOI). .. Cells were seeded in a 96-well plate, grown up to confluence and then treated with rfhSP-D (100 μg/ml) for 20 min, before inoculation with 100 TCID50 R5 tropic HIV-1JR−CSF at 37°C for 24 h. Total RNA was isolated using Trizol (Invitrogen) and the quality of RNA was assessed by nano-spectrophotometry and the nucleotide: protein ratio (260:280) was determined.

    Derivative Assay:

    Article Title: Arabidopsis UEV1D Promotes Lysine-63-Linked Polyubiquitination and Is Involved in DNA Damage Response [W]
    Article Snippet: To clone At UEV1 s, total RNA was isolated from Arabidopsis seedlings using TRIzol reagent (Invitrogen) for RT-PCR with the ThermoScript RT-PCR kit (Invitrogen) according to the manufacturer's instructions. .. Each At UEV1 ORF was amplified by PCR from the above cDNA preparation using gene-specific primers (see Supplemental Table 2 online), and the flanking Sal I restriction site was used to clone the PCR products into the yeast two-hybrid vector pGAD424E (for Gal4AD fusion), which was derived from pGAD424 , with a 1-bp frameshift at the multiple cloning site.

    Transfection:

    Article Title: A MicroRNA196a2* and TP63 Circuit Regulated by Estrogen Receptor-? and ERK2 that Controls Breast Cancer Proliferation and Invasiveness Properties
    Article Snippet: Medium was changed on day 2 and 4 of culture, and then cells were transfected with 20 nM of siGENOME Ctrl, siERα or siERK2 as described previously [ ] using Dharmafect (Dharmacon Inc, Lafayette, CO). .. For the analysis of miRNA expression, total RNA was isolated, reverse transcribed using primers specific for each miRNA (Applied Biosystems, Foster, CA) and analyzed by real-time PCR using Taqman chemistry and primers from Applied Biosystems.

    Northern Blot:

    Article Title: Choline Induces Transcriptional Repression and Proteasomal Degradation of the Malarial Phosphoethanolamine Methyltransferase ▿
    Article Snippet: Paragraph title: Real-time PCR and Northern blot analyses. ... For real time reverse transcription-PCR (RT-PCR) analysis, total RNA was extracted from the parasite pellets by using the TRIzol reagent (Invitrogen) following the manufacturer's instructions.

    Cell Culture:

    Article Title: A MicroRNA196a2* and TP63 Circuit Regulated by Estrogen Receptor-? and ERK2 that Controls Breast Cancer Proliferation and Invasiveness Properties
    Article Snippet: Paragraph title: Cell Culture, RNA Extraction, and Real-Time PCR Analysis ... For the analysis of miRNA expression, total RNA was isolated, reverse transcribed using primers specific for each miRNA (Applied Biosystems, Foster, CA) and analyzed by real-time PCR using Taqman chemistry and primers from Applied Biosystems.

    Article Title: Choline Induces Transcriptional Repression and Proteasomal Degradation of the Malarial Phosphoethanolamine Methyltransferase ▿
    Article Snippet: The parasites were resuspended in fresh medium, split into groups with equal starting parasitemia (2%), and cultured for one generation in complete RPMI 1640 medium with 0, 25, 50, 100, 200, 500, or 1,000 μM choline chloride. .. For real time reverse transcription-PCR (RT-PCR) analysis, total RNA was extracted from the parasite pellets by using the TRIzol reagent (Invitrogen) following the manufacturer's instructions.

    TaqMan microRNA Assay:

    Article Title: Star-PAP regulates tumor protein D52 through modulating miR-449a/34a in breast cancer
    Article Snippet: .. For detection of mature miR-449a/34a, total RNA was subjected to reverse TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems), and TaqMan MicroRNA Assay Kit (Applied Biosystems) was used for qPCR analysis. ..

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Arabidopsis UEV1D Promotes Lysine-63-Linked Polyubiquitination and Is Involved in DNA Damage Response [W]
    Article Snippet: .. To clone At UEV1 s, total RNA was isolated from Arabidopsis seedlings using TRIzol reagent (Invitrogen) for RT-PCR with the ThermoScript RT-PCR kit (Invitrogen) according to the manufacturer's instructions. .. Each At UEV1 ORF was amplified by PCR from the above cDNA preparation using gene-specific primers (see Supplemental Table 2 online), and the flanking Sal I restriction site was used to clone the PCR products into the yeast two-hybrid vector pGAD424E (for Gal4AD fusion), which was derived from pGAD424 , with a 1-bp frameshift at the multiple cloning site.

    Article Title: Star-PAP regulates tumor protein D52 through modulating miR-449a/34a in breast cancer
    Article Snippet: Paragraph title: qPCR and RT-PCR assay ... For detection of mature miR-449a/34a, total RNA was subjected to reverse TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems), and TaqMan MicroRNA Assay Kit (Applied Biosystems) was used for qPCR analysis.

    Article Title: Choline Induces Transcriptional Repression and Proteasomal Degradation of the Malarial Phosphoethanolamine Methyltransferase ▿
    Article Snippet: .. For real time reverse transcription-PCR (RT-PCR) analysis, total RNA was extracted from the parasite pellets by using the TRIzol reagent (Invitrogen) following the manufacturer's instructions. .. Specifically, 1 μg of total RNA was treated with DNase I (Invitrogen) to remove any contaminating genomic DNA and reverse transcribed.

    Generated:

    Article Title: Antiviral potential of human IFN-α subtypes against influenza A H3N2 infection in human lung explants reveals subtype-specific activities
    Article Snippet: .. The RNA sequencing library was generated from 500 ng total RNA using Dynabeads mRNA DIRECT Micro Purification Kit (Thermo Fisher Scientific, USA), for mRNA purification, followed by NEBNext Ultra II Directional RNA Library Prep Kit (New England BioLabs, USA), according to manufacturer’s protocols. .. The libraries were sequenced on Illumina NovaSeq 6000 using NovaSeq 6000 S1 Reagent Kit (200 and 300 cycles, paired end runs) with an average of 7.9 × 107 reads per sample.

    Article Title: Socs3 maintains the specificity of biological responses to cytokine signals during granulocyte and macrophage differentiation
    Article Snippet: .. To validate the differential expression of genes identified by microarray analysis, two independent Lin− Kit+ hematopoietic progenitor cell samples were treated with 100 ng/mL IL-6, 10 ng/mL G-CSF or saline for 4 h, and total RNA was prepared using TRIzol and RNeasy columns. cDNA was generated using Superscript II reverse transcriptase (Invitrogen, CA, USA) according to the manufacturer's instructions. .. Quantitative real-time PCR was performed using an Applied Biosystems GeneCard and a 7900HT scanner (Applied Biosystems, CA, USA).

    Transmission Assay:

    Article Title: Surfactant Protein D Reverses the Gene Signature of Transepithelial HIV-1 Passage and Restricts the Viral Transfer Across the Vaginal Barrier
    Article Snippet: Validation by Real Time RT-PCR Since, EpiVaginal tissues (of ectocervical origin) used in the assays were sufficient enough for microarray analysis, we carried out the validation of microarray data using an ectocervical cell line (Ect1/E6E7) under conditions similar to the ex vivo model of HIV-1 transmission (same MOI). .. Cells were seeded in a 96-well plate, grown up to confluence and then treated with rfhSP-D (100 μg/ml) for 20 min, before inoculation with 100 TCID50 R5 tropic HIV-1JR−CSF at 37°C for 24 h. Total RNA was isolated using Trizol (Invitrogen) and the quality of RNA was assessed by nano-spectrophotometry and the nucleotide: protein ratio (260:280) was determined.

    Sequencing:

    Article Title: SRPX2 regulates colon cancer cell metabolism by miR-192/215 via PI3K-Akt
    Article Snippet: Total RNA and miRNA was extracted from the colon cancer cells using Trizol (Invitrogen, CA, USA) based on the protocol. .. For mRNA and miRNA expression detection, real time RT-PCR was performed usinga sequence detector (ABI-Prism, Applied Biosystems) following the instructions.

    Article Title: Specific and Efficient Regression of Cancers Harboring KRAS Mutation by Targeted RNA Replacement
    Article Snippet: To analyze the level of ribozyme RNA in adenovirus-infected cells or tumor tissues from mice, total RNA was isolated with TriZol (Invitrogen), supplemented with 20 mM EDTA, and reverse-transcribed with an HSV-TK -specific primer (5′-AGTTAGCCTCCCCCATCTC-3′) in the presence of 10 mM L-argininamide. .. The cDNAs were amplified with a 5′ primer specific to the 5′ end of the KRAS RNA (5′-ATGACTGAATATAAACTTGTGGTAGTTGGA-3′) and a 3′ primer specific to the 3′ exon F. luci (5′-TCGCGGGCGCAACTGCAACTCCGATAAATA-3′) or HSV-TK sequence (5′-GTGTAGATGTTCGCGATTGTCTCGGAAG-3′).

    Recombinant:

    Article Title: Choline Induces Transcriptional Repression and Proteasomal Degradation of the Malarial Phosphoethanolamine Methyltransferase ▿
    Article Snippet: For real time reverse transcription-PCR (RT-PCR) analysis, total RNA was extracted from the parasite pellets by using the TRIzol reagent (Invitrogen) following the manufacturer's instructions. .. Serial dilutions of recombinant PCR 2.1 plasmids carrying a 470-bp, 540-bp, and 500-bp fragment of the Pf PMT , Pf TPX1 (PF14_0368 in PlasmoDB 4.4), and Pf RAB6 (PF11_0461 in PlasmoDB 4.4) genes, respectively, were used as quantification standards.

    In Vivo:

    Article Title: Specific and Efficient Regression of Cancers Harboring KRAS Mutation by Targeted RNA Replacement
    Article Snippet: Paragraph title: RNA Analysis in Cells and In Vivo ... To analyze the level of ribozyme RNA in adenovirus-infected cells or tumor tissues from mice, total RNA was isolated with TriZol (Invitrogen), supplemented with 20 mM EDTA, and reverse-transcribed with an HSV-TK -specific primer (5′-AGTTAGCCTCCCCCATCTC-3′) in the presence of 10 mM L-argininamide.

    RNA Sequencing Assay:

    Article Title: Antiviral potential of human IFN-α subtypes against influenza A H3N2 infection in human lung explants reveals subtype-specific activities
    Article Snippet: .. The RNA sequencing library was generated from 500 ng total RNA using Dynabeads mRNA DIRECT Micro Purification Kit (Thermo Fisher Scientific, USA), for mRNA purification, followed by NEBNext Ultra II Directional RNA Library Prep Kit (New England BioLabs, USA), according to manufacturer’s protocols. .. The libraries were sequenced on Illumina NovaSeq 6000 using NovaSeq 6000 S1 Reagent Kit (200 and 300 cycles, paired end runs) with an average of 7.9 × 107 reads per sample.

    Multiple Displacement Amplification:

    Article Title: A MicroRNA196a2* and TP63 Circuit Regulated by Estrogen Receptor-? and ERK2 that Controls Breast Cancer Proliferation and Invasiveness Properties
    Article Snippet: MCF-7 and MDA-MB-231 breast cancer cells were routinely maintained in Minimal Essential Medium (Sigma-Aldrich., St. Louis, MO) supplemented with 5% calf serum (HyClone, Logan, UT) or in L-15 (ATCC, Manassas, VA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT), respectively [ , ]. .. For the analysis of miRNA expression, total RNA was isolated, reverse transcribed using primers specific for each miRNA (Applied Biosystems, Foster, CA) and analyzed by real-time PCR using Taqman chemistry and primers from Applied Biosystems.

    Isolation:

    Article Title: Arabidopsis UEV1D Promotes Lysine-63-Linked Polyubiquitination and Is Involved in DNA Damage Response [W]
    Article Snippet: .. To clone At UEV1 s, total RNA was isolated from Arabidopsis seedlings using TRIzol reagent (Invitrogen) for RT-PCR with the ThermoScript RT-PCR kit (Invitrogen) according to the manufacturer's instructions. .. Each At UEV1 ORF was amplified by PCR from the above cDNA preparation using gene-specific primers (see Supplemental Table 2 online), and the flanking Sal I restriction site was used to clone the PCR products into the yeast two-hybrid vector pGAD424E (for Gal4AD fusion), which was derived from pGAD424 , with a 1-bp frameshift at the multiple cloning site.

    Article Title: Piwi-Interacting RNA1037 Enhances Chemoresistance and Motility in Human Oral Squamous Cell Carcinoma Cells
    Article Snippet: Paragraph title: RNA Isolation and Real-Time PCR ... For the quantitative assessment of XIAP mRNA expression, total RNA from the cells was extracted using Trizol (Invitrogen, USA).

    Article Title: Surfactant Protein D Reverses the Gene Signature of Transepithelial HIV-1 Passage and Restricts the Viral Transfer Across the Vaginal Barrier
    Article Snippet: .. Cells were seeded in a 96-well plate, grown up to confluence and then treated with rfhSP-D (100 μg/ml) for 20 min, before inoculation with 100 TCID50 R5 tropic HIV-1JR−CSF at 37°C for 24 h. Total RNA was isolated using Trizol (Invitrogen) and the quality of RNA was assessed by nano-spectrophotometry and the nucleotide: protein ratio (260:280) was determined. .. 1–3 μg of RNA was reverse transcribed into cDNA using Superscript III first strand synthesis kit (Invitrogen).

    Article Title: A MicroRNA196a2* and TP63 Circuit Regulated by Estrogen Receptor-? and ERK2 that Controls Breast Cancer Proliferation and Invasiveness Properties
    Article Snippet: .. For the analysis of miRNA expression, total RNA was isolated, reverse transcribed using primers specific for each miRNA (Applied Biosystems, Foster, CA) and analyzed by real-time PCR using Taqman chemistry and primers from Applied Biosystems. .. Total RNA was isolated using Trizol reagent.

    Article Title: Transcriptome Analysis Reveals New Insights into the Modulation of Endometrial Stromal Cell Receptive Phenotype by Embryo-Derived Signals Interleukin-1 and Human Chorionic Gonadotropin: Possible Involvement in Early Embryo Implantation
    Article Snippet: .. Gene expression profile for all experiments Total RNA was isolated from primary human ESCs treated with hCG (100 ng/mL) for 24 h before being stimulated or not with IL1B (0.1 ng/mL) for additional 24 h. RNA from all treated groups was compared by micro-array with the corresponding non-treated control using Affymetrix GeneChip Human Genome. ..

    Article Title: Adaptations to chronic rapamycin in mice
    Article Snippet: Colon and small intestine Frozen tissues (10–35 mg) were cryofractured as previously described , and total RNA was extracted using a mirVana miRNA Isolation Kit (Life Technologies). .. Respective cDNAs were then synthesized from 2 µg total RNA using ABI's TaqMan RNA-to-Ct Kit, and qRT-PCR was performed on 25 ng cDNA in a 10 µl reaction (2.5 ng for 18S only) using single tube inventoried TaqMan probes and TaqMan Gene Expression Master Mix (Life Technologies).

    Article Title: ERG Induces Epigenetic Activation of Tudor Domain-Containing Protein 1 (TDRD1) in ERG Rearrangement-Positive Prostate Cancer
    Article Snippet: RNA Extraction and Reverse Transcription Total RNA was isolated from exponentially growing cell lines using RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instruction. cDNA synthesis was performend using SuperScript III reverse transcriptase (Life Technologies) and oligo-dT primers (Sigma-Aldrich) following manufacturers’ instructions. .. For the measurement of LINE1-ORF2 mRNA, total RNA was treated with Turbo DNase (Life Technologies) to remove the contaminating genomic DNA.

    Article Title: Specific and Efficient Regression of Cancers Harboring KRAS Mutation by Targeted RNA Replacement
    Article Snippet: .. To analyze the level of ribozyme RNA in adenovirus-infected cells or tumor tissues from mice, total RNA was isolated with TriZol (Invitrogen), supplemented with 20 mM EDTA, and reverse-transcribed with an HSV-TK -specific primer (5′-AGTTAGCCTCCCCCATCTC-3′) in the presence of 10 mM L-argininamide. .. The cDNA was amplified with HSV-TK -specific primers (5′-TGACTTACTGGCAGGTGCTG-3′ and 5′-CCATTGTTATCTGGGCGCTTG-3′).

    Purification:

    Article Title: Antiviral potential of human IFN-α subtypes against influenza A H3N2 infection in human lung explants reveals subtype-specific activities
    Article Snippet: .. The RNA sequencing library was generated from 500 ng total RNA using Dynabeads mRNA DIRECT Micro Purification Kit (Thermo Fisher Scientific, USA), for mRNA purification, followed by NEBNext Ultra II Directional RNA Library Prep Kit (New England BioLabs, USA), according to manufacturer’s protocols. .. The libraries were sequenced on Illumina NovaSeq 6000 using NovaSeq 6000 S1 Reagent Kit (200 and 300 cycles, paired end runs) with an average of 7.9 × 107 reads per sample.

    Article Title: ERG Induces Epigenetic Activation of Tudor Domain-Containing Protein 1 (TDRD1) in ERG Rearrangement-Positive Prostate Cancer
    Article Snippet: For the measurement of LINE1-ORF2 mRNA, total RNA was treated with Turbo DNase (Life Technologies) to remove the contaminating genomic DNA. .. DNase-treated RNA was then purified using RNeasy MinElute Cleanup Kit (Qiagen) and subjected to reverse transcription using RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas, Burlington, Canada) and random hexamer primers.

    Polymerase Chain Reaction:

    Article Title: Arabidopsis UEV1D Promotes Lysine-63-Linked Polyubiquitination and Is Involved in DNA Damage Response [W]
    Article Snippet: To clone At UEV1 s, total RNA was isolated from Arabidopsis seedlings using TRIzol reagent (Invitrogen) for RT-PCR with the ThermoScript RT-PCR kit (Invitrogen) according to the manufacturer's instructions. .. Each At UEV1 ORF was amplified by PCR from the above cDNA preparation using gene-specific primers (see Supplemental Table 2 online), and the flanking Sal I restriction site was used to clone the PCR products into the yeast two-hybrid vector pGAD424E (for Gal4AD fusion), which was derived from pGAD424 , with a 1-bp frameshift at the multiple cloning site.

    Article Title: SRPX2 regulates colon cancer cell metabolism by miR-192/215 via PI3K-Akt
    Article Snippet: Paragraph title: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) ... Total RNA and miRNA was extracted from the colon cancer cells using Trizol (Invitrogen, CA, USA) based on the protocol.

    Article Title: Choline Induces Transcriptional Repression and Proteasomal Degradation of the Malarial Phosphoethanolamine Methyltransferase ▿
    Article Snippet: For real time reverse transcription-PCR (RT-PCR) analysis, total RNA was extracted from the parasite pellets by using the TRIzol reagent (Invitrogen) following the manufacturer's instructions. .. Serial dilutions of recombinant PCR 2.1 plasmids carrying a 470-bp, 540-bp, and 500-bp fragment of the Pf PMT , Pf TPX1 (PF14_0368 in PlasmoDB 4.4), and Pf RAB6 (PF11_0461 in PlasmoDB 4.4) genes, respectively, were used as quantification standards.

    Mouse Assay:

    Article Title: Specific and Efficient Regression of Cancers Harboring KRAS Mutation by Targeted RNA Replacement
    Article Snippet: .. To analyze the level of ribozyme RNA in adenovirus-infected cells or tumor tissues from mice, total RNA was isolated with TriZol (Invitrogen), supplemented with 20 mM EDTA, and reverse-transcribed with an HSV-TK -specific primer (5′-AGTTAGCCTCCCCCATCTC-3′) in the presence of 10 mM L-argininamide. .. The cDNA was amplified with HSV-TK -specific primers (5′-TGACTTACTGGCAGGTGCTG-3′ and 5′-CCATTGTTATCTGGGCGCTTG-3′).

    Plasmid Preparation:

    Article Title: Arabidopsis UEV1D Promotes Lysine-63-Linked Polyubiquitination and Is Involved in DNA Damage Response [W]
    Article Snippet: Paragraph title: Cloning Arabidopsis UEV1 cDNAs and Plasmid Construction ... To clone At UEV1 s, total RNA was isolated from Arabidopsis seedlings using TRIzol reagent (Invitrogen) for RT-PCR with the ThermoScript RT-PCR kit (Invitrogen) according to the manufacturer's instructions.

    Real-time Polymerase Chain Reaction:

    Article Title: Piwi-Interacting RNA1037 Enhances Chemoresistance and Motility in Human Oral Squamous Cell Carcinoma Cells
    Article Snippet: Paragraph title: RNA Isolation and Real-Time PCR ... For the quantitative assessment of XIAP mRNA expression, total RNA from the cells was extracted using Trizol (Invitrogen, USA).

    Article Title: Surfactant Protein D Reverses the Gene Signature of Transepithelial HIV-1 Passage and Restricts the Viral Transfer Across the Vaginal Barrier
    Article Snippet: Cells were seeded in a 96-well plate, grown up to confluence and then treated with rfhSP-D (100 μg/ml) for 20 min, before inoculation with 100 TCID50 R5 tropic HIV-1JR−CSF at 37°C for 24 h. Total RNA was isolated using Trizol (Invitrogen) and the quality of RNA was assessed by nano-spectrophotometry and the nucleotide: protein ratio (260:280) was determined. .. The resulting cDNA was used for real time PCR via the Bio-Rad CFX96 TouchTM real-time PCR detection system using the iQTM SYBR Green Supermix (Bio-Rad, USA).

    Article Title: A MicroRNA196a2* and TP63 Circuit Regulated by Estrogen Receptor-? and ERK2 that Controls Breast Cancer Proliferation and Invasiveness Properties
    Article Snippet: .. For the analysis of miRNA expression, total RNA was isolated, reverse transcribed using primers specific for each miRNA (Applied Biosystems, Foster, CA) and analyzed by real-time PCR using Taqman chemistry and primers from Applied Biosystems. .. Total RNA was isolated using Trizol reagent.

    Article Title: Star-PAP regulates tumor protein D52 through modulating miR-449a/34a in breast cancer
    Article Snippet: .. For detection of mature miR-449a/34a, total RNA was subjected to reverse TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems), and TaqMan MicroRNA Assay Kit (Applied Biosystems) was used for qPCR analysis. ..

    Article Title: TNF-alpha upregulates the A2B adenosine receptor gene: the role of NAD(P)H oxidase 4
    Article Snippet: Total RNA from aortic VSMC was prepared with Trizol® (Invitrogen, Carlsbad, Ca) according to the manufacturer’s instructions [ ]. .. A2B AR mRNA was quantified using ABI Gene Array TaqMan® primers (ABI, Foster City, CA, cat# Rn00567696_m1), and 18s rRNA (cat# 4319413E) with the TaqMan® Gene Expression Master Mix (ABI, Foster City, CA, cat# 4370048), using the ABI 7300 Real-Time PCR System.

    Article Title: Socs3 maintains the specificity of biological responses to cytokine signals during granulocyte and macrophage differentiation
    Article Snippet: Paragraph title: Quantitative real-time PCR ... To validate the differential expression of genes identified by microarray analysis, two independent Lin− Kit+ hematopoietic progenitor cell samples were treated with 100 ng/mL IL-6, 10 ng/mL G-CSF or saline for 4 h, and total RNA was prepared using TRIzol and RNeasy columns. cDNA was generated using Superscript II reverse transcriptase (Invitrogen, CA, USA) according to the manufacturer's instructions.

    Article Title: Choline Induces Transcriptional Repression and Proteasomal Degradation of the Malarial Phosphoethanolamine Methyltransferase ▿
    Article Snippet: Paragraph title: Real-time PCR and Northern blot analyses. ... For real time reverse transcription-PCR (RT-PCR) analysis, total RNA was extracted from the parasite pellets by using the TRIzol reagent (Invitrogen) following the manufacturer's instructions.

    RNA Extraction:

    Article Title: A MicroRNA196a2* and TP63 Circuit Regulated by Estrogen Receptor-? and ERK2 that Controls Breast Cancer Proliferation and Invasiveness Properties
    Article Snippet: Paragraph title: Cell Culture, RNA Extraction, and Real-Time PCR Analysis ... For the analysis of miRNA expression, total RNA was isolated, reverse transcribed using primers specific for each miRNA (Applied Biosystems, Foster, CA) and analyzed by real-time PCR using Taqman chemistry and primers from Applied Biosystems.

    Article Title: ERG Induces Epigenetic Activation of Tudor Domain-Containing Protein 1 (TDRD1) in ERG Rearrangement-Positive Prostate Cancer
    Article Snippet: Paragraph title: RNA Extraction and Reverse Transcription ... For the measurement of LINE1-ORF2 mRNA, total RNA was treated with Turbo DNase (Life Technologies) to remove the contaminating genomic DNA.

    Agarose Gel Electrophoresis:

    Article Title: Star-PAP regulates tumor protein D52 through modulating miR-449a/34a in breast cancer
    Article Snippet: For RT-PCR, PrimeSTAR HS DNA Polymerase (Takara, Kusatsu, Japan) was used, and the products were analyzed in agarose gel. .. For detection of mature miR-449a/34a, total RNA was subjected to reverse TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems), and TaqMan MicroRNA Assay Kit (Applied Biosystems) was used for qPCR analysis.

    Article Title: Anomalous electrophoretic migration of newly synthesized ribosomal RNAs and their precursors from cells with DKC1 mutations
    Article Snippet: Paragraph title: Agarose gel electrophoresis ... Total RNA was extracted from ES cells and MEF cells by using TRIzol Reagent (Invitrogen, Carlsbad, CA).

    Electrophoresis:

    Article Title: Anomalous electrophoretic migration of newly synthesized ribosomal RNAs and their precursors from cells with DKC1 mutations
    Article Snippet: Total RNA was extracted from ES cells and MEF cells by using TRIzol Reagent (Invitrogen, Carlsbad, CA). .. Total RNA was separated on 1.25% agarose formaldehyde (2.2M) gel using 1× MOPS electrophoresis running buffer (0.02M MOPS, 0.005M Sodium Acetate, 0.001M EDTA and 0.001M EGTA).

    Transgenic Assay:

    Article Title: Choline Induces Transcriptional Repression and Proteasomal Degradation of the Malarial Phosphoethanolamine Methyltransferase ▿
    Article Snippet: Wild-type 3D7 and transgenic parasites constitutively expressing Pf PMT under the calmodulin promoter (TP-CAM-PfPMT) were synchronized with 5% d -sorbitol and grown to early trophozoite stage in medium without choline. .. For real time reverse transcription-PCR (RT-PCR) analysis, total RNA was extracted from the parasite pellets by using the TRIzol reagent (Invitrogen) following the manufacturer's instructions.

    Random Hexamer Labeling:

    Article Title: ERG Induces Epigenetic Activation of Tudor Domain-Containing Protein 1 (TDRD1) in ERG Rearrangement-Positive Prostate Cancer
    Article Snippet: For the measurement of LINE1-ORF2 mRNA, total RNA was treated with Turbo DNase (Life Technologies) to remove the contaminating genomic DNA. .. DNase-treated RNA was then purified using RNeasy MinElute Cleanup Kit (Qiagen) and subjected to reverse transcription using RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas, Burlington, Canada) and random hexamer primers.

    Concentration Assay:

    Article Title: Anomalous electrophoretic migration of newly synthesized ribosomal RNAs and their precursors from cells with DKC1 mutations
    Article Snippet: Total RNA was extracted from ES cells and MEF cells by using TRIzol Reagent (Invitrogen, Carlsbad, CA). .. RNA was mixed with 2X volumes RNA Sample Loading Buffer (Deionized formamide 62.5% (v/v), formaldehyde 1.14 M, bromphenol blue 200 μg/mL, xylene cyanol 200 μg/mL, MOPS-EDTA-sodium acetate at 1.25× working concentration.) and denatured for 1, 5 or 20 minutes at 65°C.

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    The highest quality RNA available from Ambion it is DNase treated and subjected to unsurpassed quality control standards One tube containing 100 µg is provided at a concentration of 1
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    Thermo Fisher quantitative rt pcr analysis total rna
    Stable forced SUMO1 expression enhanced the colony formation, proliferation, migration, cell cycle progression, and invasion of A549 cells in vitro. ( a ) Detection of messenger <t>RNA</t> (mRNA) expression of SUMO1 in different lung cancer cell lines by quantitative real time <t>(qRT)‐PCR.</t> ( b ) Similar results were obtained through Western blot analysis. ( c ) qRT‐PCR analysis revealed that SUMO1 mRNA expression levels were increased in SUMO1 overexpressed A549 cells compared to control cells. ( d ) Similar results were obtained through Western blot analysis (passages 15 and 30). Upregulation of SUMO1 enhanced the ( e , f ) colony‐formation ability, ( g ) proliferation, ( h , i ) migration, and ( k , l ) invasion of A549 cells. ( j ) Forced expression of SUMO1 increased the number of A549 cells in the S phase of the cell cycle. * P
    Quantitative Rt Pcr Analysis Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quantitative rt pcr analysis total rna/product/Thermo Fisher
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    Thermo Fisher total rna
    Demethylation of hemi-methylated CpG sites up-regulates pluripotent genes. a – c Approximately 20,000 protein-coding genes were divided into different groups based on their methylation levels and CpG densities near TSS (−1.5 ~ + 2.0 kb). The number of genes in the different groups were summarized in a . Genes with 42–83 CpG sites near their TSS (−1.5 ~ + 2.0 kb) were highlighted as in Zone I in a . Of the genes in Zone I, the genes with methylation levels between 40 and 60% were highlighted in Zone II. The changes in methylation of approximately 14,500 genes detected in the current RRBS were summarized in b , while the changes in the expression of approximately 11,000 genes detected in the current <t>RNA-seq</t> were summarized in c . Groups with less than 50 genes detected in RRBS or RNA-seq were not listed in b and c . d – f Demethylation of genes highlighted in Zone I and Zone II in a were compared to the average demethylation of all genes in d , e . The expression changes of 698 genes in Zone II were compared to the expression changes of all genes ( f ). g – i GO analysis results of the 698 genes in Zone II ( g ). The 22 genes that undergo significant up-regulation were selected and their demethylation was listed in h . Of these 22 genes, the expression changes of 6 pluripotent genes were determined by <t>qPCR</t> ( i )
    Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 277 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher quantitative real time polymerase chain reaction qrt pcr total rna
    Up-regulation of miR-96 and down-regulation of FOXO1 in tumor tissues and HepG2 cells. a miR-96 relative expression in tumor tissues and non-tumor tissues by <t>qRT-PCR;</t> b FOXO1 mRNA relative expression in tumor tissues and non-tumor tissues by qRT-PCR; c FOXO1 protein relative expression in tumor tissues and non-tumor tissues by Western blot; d miR-96 relative expression in L02 and HepG2 cells by qRT-PCR; e FOXO1 mRNA relative expression in L02 and HepG2 cells by qRT-PCR; f FOXO1 protein relative expression in L02 and HepG2 cells by Western blot. ** P
    Quantitative Real Time Polymerase Chain Reaction Qrt Pcr Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 364 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quantitative real time polymerase chain reaction qrt pcr total rna/product/Thermo Fisher
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    Image Search Results


    Stable forced SUMO1 expression enhanced the colony formation, proliferation, migration, cell cycle progression, and invasion of A549 cells in vitro. ( a ) Detection of messenger RNA (mRNA) expression of SUMO1 in different lung cancer cell lines by quantitative real time (qRT)‐PCR. ( b ) Similar results were obtained through Western blot analysis. ( c ) qRT‐PCR analysis revealed that SUMO1 mRNA expression levels were increased in SUMO1 overexpressed A549 cells compared to control cells. ( d ) Similar results were obtained through Western blot analysis (passages 15 and 30). Upregulation of SUMO1 enhanced the ( e , f ) colony‐formation ability, ( g ) proliferation, ( h , i ) migration, and ( k , l ) invasion of A549 cells. ( j ) Forced expression of SUMO1 increased the number of A549 cells in the S phase of the cell cycle. * P

    Journal: Thoracic Cancer

    Article Title: SUMO1 promotes the proliferation and invasion of non‐small cell lung cancer cells by regulating NF‐κB

    doi: 10.1111/1759-7714.12895

    Figure Lengend Snippet: Stable forced SUMO1 expression enhanced the colony formation, proliferation, migration, cell cycle progression, and invasion of A549 cells in vitro. ( a ) Detection of messenger RNA (mRNA) expression of SUMO1 in different lung cancer cell lines by quantitative real time (qRT)‐PCR. ( b ) Similar results were obtained through Western blot analysis. ( c ) qRT‐PCR analysis revealed that SUMO1 mRNA expression levels were increased in SUMO1 overexpressed A549 cells compared to control cells. ( d ) Similar results were obtained through Western blot analysis (passages 15 and 30). Upregulation of SUMO1 enhanced the ( e , f ) colony‐formation ability, ( g ) proliferation, ( h , i ) migration, and ( k , l ) invasion of A549 cells. ( j ) Forced expression of SUMO1 increased the number of A549 cells in the S phase of the cell cycle. * P

    Article Snippet: Quantitative RT‐PCR analysis Total RNA was isolated using the TRIzol reagent according to the manufacturer's protocol (Thermo Fisher Scientific).

    Techniques: Expressing, Migration, In Vitro, Quantitative RT-PCR, Western Blot

    Downregulation of SUMO1 suppresses the proliferation, cell cycle progression, colony formation, migration, and invasion of Calu‐1 cells in vitro. ( a ) Quantitative real time‐PCR analysis revealed that the messenger RNA (mRNA) expression levels of SUMO1 in short hairpin RNA (shRNA)‐SUMO1‐1 Calu‐1 cells were significantly downregulated. ( b ) Similar results were obtained through Western blot analysis. ( c ) Downregulation of SUMO1 expression significantly inhibited the ( c ) proliferation, ( e , f ) colony‐formation ability, ( g , h ) migration, and ( i , j ) invasion of Calu‐1 cells. ( d ) Downregulation of SUMO1 reduced the number of Calu‐1 cells in the S phase of the cell cycle.

    Journal: Thoracic Cancer

    Article Title: SUMO1 promotes the proliferation and invasion of non‐small cell lung cancer cells by regulating NF‐κB

    doi: 10.1111/1759-7714.12895

    Figure Lengend Snippet: Downregulation of SUMO1 suppresses the proliferation, cell cycle progression, colony formation, migration, and invasion of Calu‐1 cells in vitro. ( a ) Quantitative real time‐PCR analysis revealed that the messenger RNA (mRNA) expression levels of SUMO1 in short hairpin RNA (shRNA)‐SUMO1‐1 Calu‐1 cells were significantly downregulated. ( b ) Similar results were obtained through Western blot analysis. ( c ) Downregulation of SUMO1 expression significantly inhibited the ( c ) proliferation, ( e , f ) colony‐formation ability, ( g , h ) migration, and ( i , j ) invasion of Calu‐1 cells. ( d ) Downregulation of SUMO1 reduced the number of Calu‐1 cells in the S phase of the cell cycle.

    Article Snippet: Quantitative RT‐PCR analysis Total RNA was isolated using the TRIzol reagent according to the manufacturer's protocol (Thermo Fisher Scientific).

    Techniques: Migration, In Vitro, Real-time Polymerase Chain Reaction, Expressing, shRNA, Western Blot

    Demethylation of hemi-methylated CpG sites up-regulates pluripotent genes. a – c Approximately 20,000 protein-coding genes were divided into different groups based on their methylation levels and CpG densities near TSS (−1.5 ~ + 2.0 kb). The number of genes in the different groups were summarized in a . Genes with 42–83 CpG sites near their TSS (−1.5 ~ + 2.0 kb) were highlighted as in Zone I in a . Of the genes in Zone I, the genes with methylation levels between 40 and 60% were highlighted in Zone II. The changes in methylation of approximately 14,500 genes detected in the current RRBS were summarized in b , while the changes in the expression of approximately 11,000 genes detected in the current RNA-seq were summarized in c . Groups with less than 50 genes detected in RRBS or RNA-seq were not listed in b and c . d – f Demethylation of genes highlighted in Zone I and Zone II in a were compared to the average demethylation of all genes in d , e . The expression changes of 698 genes in Zone II were compared to the expression changes of all genes ( f ). g – i GO analysis results of the 698 genes in Zone II ( g ). The 22 genes that undergo significant up-regulation were selected and their demethylation was listed in h . Of these 22 genes, the expression changes of 6 pluripotent genes were determined by qPCR ( i )

    Journal: Cell Discovery

    Article Title: Hemi-methylated CpG sites connect Dnmt1-knockdown-induced and Tet1-induced DNA demethylation during somatic cell reprogramming

    doi: 10.1038/s41421-018-0074-6

    Figure Lengend Snippet: Demethylation of hemi-methylated CpG sites up-regulates pluripotent genes. a – c Approximately 20,000 protein-coding genes were divided into different groups based on their methylation levels and CpG densities near TSS (−1.5 ~ + 2.0 kb). The number of genes in the different groups were summarized in a . Genes with 42–83 CpG sites near their TSS (−1.5 ~ + 2.0 kb) were highlighted as in Zone I in a . Of the genes in Zone I, the genes with methylation levels between 40 and 60% were highlighted in Zone II. The changes in methylation of approximately 14,500 genes detected in the current RRBS were summarized in b , while the changes in the expression of approximately 11,000 genes detected in the current RNA-seq were summarized in c . Groups with less than 50 genes detected in RRBS or RNA-seq were not listed in b and c . d – f Demethylation of genes highlighted in Zone I and Zone II in a were compared to the average demethylation of all genes in d , e . The expression changes of 698 genes in Zone II were compared to the expression changes of all genes ( f ). g – i GO analysis results of the 698 genes in Zone II ( g ). The 22 genes that undergo significant up-regulation were selected and their demethylation was listed in h . Of these 22 genes, the expression changes of 6 pluripotent genes were determined by qPCR ( i )

    Article Snippet: Quantitative RT-PCR (qPCR) For the qPCR, total RNA was extracted from the cells using TRIzol (Thermo Fisher), and 5 μg of RNA were used to synthesize cDNA with ReverTra Ace® (Toyobo) and oligo-dT (Takara) according to the manufacturers’ instructions.

    Techniques: Methylation, Expressing, RNA Sequencing Assay, Real-time Polymerase Chain Reaction

    Two types of DNA demethylation differentially influence gene expression. The results obtained from the WGBS, ATAC-seq, and RNA-seq in assays of MEFs were analyzed together in a – g . The results obtained from WGBS (MEFs) and RRBS/RNA-seq (control, Tet1, and sh-Dnmt1 groups in MEFs) were analyzed in h , i . a First, genes were divided into 102 groups based their methylation levels (0%, 0–1%, …, 99–100%, and 100%). Then, the genes in each group were equally separated into two sub-groups, i.e., high enrichment and low enrichment, based on their AMDs. The genes from all high/low enrichment sub-groups were combined and sorted based on their methylation levels, and were presented along with their chromatin accessibility. The chromatin accessibilities generated from ATAC-seq were listed near TSS (−2.0 ~ + 2.0 kb). Gene methylation was plotted on the left. b First, the genes were divided into 20 groups (0–5%, …, and 95–100%). Then, the averages of gene methylation and chromatin accessibility were plotted. c – g Six sets of genes with particular methylation levels and enrichment of hemi-methylation were selected ( c ). The average methylation levels ( d ), enrichment of hemi-methylation ( e ), chromatin accessibilities ( f ), and gene expression ( g ) in MEFs were listed. The number of genes in each group was provided. h – i Of the 300 selected genes, the expression of 197 genes was detected in the current RNA-seq. The expression changes induced by Tet1 and sh-Dnmt1 were plotted in h . In total, 20 genes were randomly selected from these 197 genes, and the chromatin accessibilities of the genes in the Tet1 and sh-Dnmt1 groups were determined by ATAC-qPCR, compared, and plotted in i

    Journal: Cell Discovery

    Article Title: Hemi-methylated CpG sites connect Dnmt1-knockdown-induced and Tet1-induced DNA demethylation during somatic cell reprogramming

    doi: 10.1038/s41421-018-0074-6

    Figure Lengend Snippet: Two types of DNA demethylation differentially influence gene expression. The results obtained from the WGBS, ATAC-seq, and RNA-seq in assays of MEFs were analyzed together in a – g . The results obtained from WGBS (MEFs) and RRBS/RNA-seq (control, Tet1, and sh-Dnmt1 groups in MEFs) were analyzed in h , i . a First, genes were divided into 102 groups based their methylation levels (0%, 0–1%, …, 99–100%, and 100%). Then, the genes in each group were equally separated into two sub-groups, i.e., high enrichment and low enrichment, based on their AMDs. The genes from all high/low enrichment sub-groups were combined and sorted based on their methylation levels, and were presented along with their chromatin accessibility. The chromatin accessibilities generated from ATAC-seq were listed near TSS (−2.0 ~ + 2.0 kb). Gene methylation was plotted on the left. b First, the genes were divided into 20 groups (0–5%, …, and 95–100%). Then, the averages of gene methylation and chromatin accessibility were plotted. c – g Six sets of genes with particular methylation levels and enrichment of hemi-methylation were selected ( c ). The average methylation levels ( d ), enrichment of hemi-methylation ( e ), chromatin accessibilities ( f ), and gene expression ( g ) in MEFs were listed. The number of genes in each group was provided. h – i Of the 300 selected genes, the expression of 197 genes was detected in the current RNA-seq. The expression changes induced by Tet1 and sh-Dnmt1 were plotted in h . In total, 20 genes were randomly selected from these 197 genes, and the chromatin accessibilities of the genes in the Tet1 and sh-Dnmt1 groups were determined by ATAC-qPCR, compared, and plotted in i

    Article Snippet: Quantitative RT-PCR (qPCR) For the qPCR, total RNA was extracted from the cells using TRIzol (Thermo Fisher), and 5 μg of RNA were used to synthesize cDNA with ReverTra Ace® (Toyobo) and oligo-dT (Takara) according to the manufacturers’ instructions.

    Techniques: Expressing, RNA Sequencing Assay, Methylation, Generated, Real-time Polymerase Chain Reaction

    Schematic illustration of the study design. Following culture of the IPF fibroblasts and healthy lung fibroblasts, the total RNA were extracted and deep sequenced using a next-generation sequencing platform. The differentially expressed genes ( > 2 fold-change) were identified, and analyzed with IPA and DAVID for pathway analysis and functional interpretation. The GEO IPF databases were analyzed to confirm dysregulated genes identified in the present study. Conversely, the differentially expressed miRNAs ( > 2 fold-change) were analyzed with miRmap for target prediction. Then, genes with potential miRNA-mRNA interactions were determined by Venn diagram analysis. These miRNA-mRNA interactions were verified by a second miRNA prediction database, TargetScan. Finally, a literature search was performed and a hypothesis was generated. IPF, idiopathic pulmonary fibrosis; IPA, Ingenuity ® Pathway Analysis; DAVID, Database for Annotation, Visualization, and Integrated Discovery; GEO, Gene Expression Omnibus.

    Journal: International Journal of Molecular Medicine

    Article Title: Bioinformatic analysis of next-generation sequencing data to identify dysregulated genes in fibroblasts of idiopathic pulmonary fibrosis

    doi: 10.3892/ijmm.2019.4086

    Figure Lengend Snippet: Schematic illustration of the study design. Following culture of the IPF fibroblasts and healthy lung fibroblasts, the total RNA were extracted and deep sequenced using a next-generation sequencing platform. The differentially expressed genes ( > 2 fold-change) were identified, and analyzed with IPA and DAVID for pathway analysis and functional interpretation. The GEO IPF databases were analyzed to confirm dysregulated genes identified in the present study. Conversely, the differentially expressed miRNAs ( > 2 fold-change) were analyzed with miRmap for target prediction. Then, genes with potential miRNA-mRNA interactions were determined by Venn diagram analysis. These miRNA-mRNA interactions were verified by a second miRNA prediction database, TargetScan. Finally, a literature search was performed and a hypothesis was generated. IPF, idiopathic pulmonary fibrosis; IPA, Ingenuity ® Pathway Analysis; DAVID, Database for Annotation, Visualization, and Integrated Discovery; GEO, Gene Expression Omnibus.

    Article Snippet: In brief, total RNA from the DHLF-IPF and NHLF cells were extracted with TRIzol® reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at Welgene Biotech Co., Ltd. (Taipei, Taiwan).

    Techniques: Next-Generation Sequencing, Indirect Immunoperoxidase Assay, Functional Assay, Generated, Expressing

    Up-regulation of miR-96 and down-regulation of FOXO1 in tumor tissues and HepG2 cells. a miR-96 relative expression in tumor tissues and non-tumor tissues by qRT-PCR; b FOXO1 mRNA relative expression in tumor tissues and non-tumor tissues by qRT-PCR; c FOXO1 protein relative expression in tumor tissues and non-tumor tissues by Western blot; d miR-96 relative expression in L02 and HepG2 cells by qRT-PCR; e FOXO1 mRNA relative expression in L02 and HepG2 cells by qRT-PCR; f FOXO1 protein relative expression in L02 and HepG2 cells by Western blot. ** P

    Journal: Cancer Cell International

    Article Title: miR-96 exerts carcinogenic effect by activating AKT/GSK-3β/β-catenin signaling pathway through targeting inhibition of FOXO1 in hepatocellular carcinoma

    doi: 10.1186/s12935-019-0756-7

    Figure Lengend Snippet: Up-regulation of miR-96 and down-regulation of FOXO1 in tumor tissues and HepG2 cells. a miR-96 relative expression in tumor tissues and non-tumor tissues by qRT-PCR; b FOXO1 mRNA relative expression in tumor tissues and non-tumor tissues by qRT-PCR; c FOXO1 protein relative expression in tumor tissues and non-tumor tissues by Western blot; d miR-96 relative expression in L02 and HepG2 cells by qRT-PCR; e FOXO1 mRNA relative expression in L02 and HepG2 cells by qRT-PCR; f FOXO1 protein relative expression in L02 and HepG2 cells by Western blot. ** P

    Article Snippet: Quantitative real time polymerase chain reaction (qRT-PCR) Total RNA in tumor tissues and cells was obtained by Trizol kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the the instruction manual.

    Techniques: Expressing, Quantitative RT-PCR, Western Blot

    FOXO1 was directly suppressed by miR-96. a Prediction of binding sites for FOXO1 and miR-96 by Target Scan; b Luciferase reporter assay; c FOXO1 mRNA expression by qRT-PCR; d FOXO1 protein expression by Western blot. ## P

    Journal: Cancer Cell International

    Article Title: miR-96 exerts carcinogenic effect by activating AKT/GSK-3β/β-catenin signaling pathway through targeting inhibition of FOXO1 in hepatocellular carcinoma

    doi: 10.1186/s12935-019-0756-7

    Figure Lengend Snippet: FOXO1 was directly suppressed by miR-96. a Prediction of binding sites for FOXO1 and miR-96 by Target Scan; b Luciferase reporter assay; c FOXO1 mRNA expression by qRT-PCR; d FOXO1 protein expression by Western blot. ## P

    Article Snippet: Quantitative real time polymerase chain reaction (qRT-PCR) Total RNA in tumor tissues and cells was obtained by Trizol kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the the instruction manual.

    Techniques: Binding Assay, Luciferase, Reporter Assay, Expressing, Quantitative RT-PCR, Western Blot