target mrna  (Thermo Fisher)


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    Structured Review

    Thermo Fisher target mrna
    Effect of oral Lactobacillus chungangensis CAU 28 cream cheese administration on <t>mRNA</t> expression. Target mRNA expression levels including those of ( a ) IL-4, ( b ) IL-5, ( c ) IL-10, (d) IL-12, (e) IFN-γ, (f) TNF-α, and (g) <t>IL-1β</t> were determined via real-time polymerase chain reaction. Significance marks (* p
    Target Mrna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 10282 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Cream Cheese-Derived Lactococcus chungangensis CAU 28 Modulates the Gut Microbiota and Alleviates Atopic Dermatitis in BALB/c Mice"

    Article Title: Cream Cheese-Derived Lactococcus chungangensis CAU 28 Modulates the Gut Microbiota and Alleviates Atopic Dermatitis in BALB/c Mice

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-36864-5

    Effect of oral Lactobacillus chungangensis CAU 28 cream cheese administration on mRNA expression. Target mRNA expression levels including those of ( a ) IL-4, ( b ) IL-5, ( c ) IL-10, (d) IL-12, (e) IFN-γ, (f) TNF-α, and (g) IL-1β were determined via real-time polymerase chain reaction. Significance marks (* p
    Figure Legend Snippet: Effect of oral Lactobacillus chungangensis CAU 28 cream cheese administration on mRNA expression. Target mRNA expression levels including those of ( a ) IL-4, ( b ) IL-5, ( c ) IL-10, (d) IL-12, (e) IFN-γ, (f) TNF-α, and (g) IL-1β were determined via real-time polymerase chain reaction. Significance marks (* p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    2) Product Images from "Treatment with a neutralizing anti-murine interleukin-17 antibody after the onset of coxsackievirus b3-induced viral myocarditis reduces myocardium inflammation"

    Article Title: Treatment with a neutralizing anti-murine interleukin-17 antibody after the onset of coxsackievirus b3-induced viral myocarditis reduces myocardium inflammation

    Journal: Virology Journal

    doi: 10.1186/1743-422X-8-17

    IL-17, IL-6 and TNF-α mRNA transcription in VMC myocardium and protein level in the serum . A. Representative images showing semiquantitative RT-PCR for IL-17, IL-6 and TNF-α transcription. 1. Normal group; 2. IL-17mAb group; 3. Isotype control group; 4. PBS group. B, Densitometric quantitation of the PCR bands showed that, among the IL-17mAb, isotype control and PBS groups, the mRNA expression did not differ with each other; C, The levels of serum IL-17 in different groups, measured by ELISA. The IL-17 protein levels were 11.81 ± 2.66, 22.56 ± 3.68, 33.63 ± 5.50, 33.68 ± 6.13 pg/ml in normal, IL-17mAb, Isotype control and PBS group respectively. D, The levels of serum IL-6 and TNF-α in different group, measured by ELISA. The IL-6 levels were 23.15 ± 8.59, 71.28.50 ± 15.80, 77.81 ± 6.54, 78.18 ± 6.26 pg/ml, and the TNF-α levels were 27.80 ± 4.52, 64.24 ± 6.71, 67.89 ± 5.25, 63.45 ± 2.71 pg/ml respectively. Normal group (n = 8), IL17mAb (n = 7), isotype contro(n = 4), and PBS group(n = 5).* P
    Figure Legend Snippet: IL-17, IL-6 and TNF-α mRNA transcription in VMC myocardium and protein level in the serum . A. Representative images showing semiquantitative RT-PCR for IL-17, IL-6 and TNF-α transcription. 1. Normal group; 2. IL-17mAb group; 3. Isotype control group; 4. PBS group. B, Densitometric quantitation of the PCR bands showed that, among the IL-17mAb, isotype control and PBS groups, the mRNA expression did not differ with each other; C, The levels of serum IL-17 in different groups, measured by ELISA. The IL-17 protein levels were 11.81 ± 2.66, 22.56 ± 3.68, 33.63 ± 5.50, 33.68 ± 6.13 pg/ml in normal, IL-17mAb, Isotype control and PBS group respectively. D, The levels of serum IL-6 and TNF-α in different group, measured by ELISA. The IL-6 levels were 23.15 ± 8.59, 71.28.50 ± 15.80, 77.81 ± 6.54, 78.18 ± 6.26 pg/ml, and the TNF-α levels were 27.80 ± 4.52, 64.24 ± 6.71, 67.89 ± 5.25, 63.45 ± 2.71 pg/ml respectively. Normal group (n = 8), IL17mAb (n = 7), isotype contro(n = 4), and PBS group(n = 5).* P

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Quantitation Assay, Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay

    3) Product Images from "Regulation of the human tyrosinase gene in retinal pigment epithelium cells: the significance of transcription factor orthodenticle homeobox 2 and its polymorphic binding site"

    Article Title: Regulation of the human tyrosinase gene in retinal pigment epithelium cells: the significance of transcription factor orthodenticle homeobox 2 and its polymorphic binding site

    Journal: Molecular Vision

    doi:

    Expression of TYR , MITF-A , MITF-D , MITF-H , MITF-M , and OTX2 mRNA in RPE cells. ARPE-19 mRNA was collected at various time points (6 h, 24 h, 48 h, 96 h, 14 days, and 30 days). Other samples were isolated at 96 h from D407 RPE cells and G-361 melanoma cells, a positive control for TYR expression. Results are presented as the mean normalized expression±SD relative to 6 h ARPE-19 culture (=1) from three to four independent cultures each performed in triplicate. Due to very low expression levels, MITF-D values were normalized relative to MITF-H (6 h ARPE-19 culture=1).
    Figure Legend Snippet: Expression of TYR , MITF-A , MITF-D , MITF-H , MITF-M , and OTX2 mRNA in RPE cells. ARPE-19 mRNA was collected at various time points (6 h, 24 h, 48 h, 96 h, 14 days, and 30 days). Other samples were isolated at 96 h from D407 RPE cells and G-361 melanoma cells, a positive control for TYR expression. Results are presented as the mean normalized expression±SD relative to 6 h ARPE-19 culture (=1) from three to four independent cultures each performed in triplicate. Due to very low expression levels, MITF-D values were normalized relative to MITF-H (6 h ARPE-19 culture=1).

    Techniques Used: Expressing, Isolation, Positive Control

    4) Product Images from "Functional and Structural Analysis of Maize Hsp101 IRES"

    Article Title: Functional and Structural Analysis of Maize Hsp101 IRES

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0107459

    Functional analysis of the maize hsp101 IRES. a) Maize Hsp101 5′UTR sequence. Arrows indicate the first nucleotide of the fragments used to identify the functional IRES region. b) Schematic representation of the bicistronic constructs pBIC-5 plasmid was used as positive control. Hsp101 fragments (sense orientation) were inserted between the chloramphenicol acetyl transferase (CAT) and luciferase (Luc) reporter genes. c) Translation efficiency of the mutant IRES elements in RRL. Equal amounts of in vitro synthesized RNAs pBIC-5, 101+1, 101Δ17 or 101Δ50 were used to program translation using Lb-untreated (-) RRL or Lb-treated (+) RRL. Proteins were resolved by SDS–PAGE and visualized by autoradiography of dry gels. Synthesis of luciferase driven by the indicated IRES elements in the presence or absence of the Lb protease is shown on the insert. The histogram shows the intensity of luciferase translation efficiency driven by the indicated RNAs in the presence of Lb (cap-independent).
    Figure Legend Snippet: Functional analysis of the maize hsp101 IRES. a) Maize Hsp101 5′UTR sequence. Arrows indicate the first nucleotide of the fragments used to identify the functional IRES region. b) Schematic representation of the bicistronic constructs pBIC-5 plasmid was used as positive control. Hsp101 fragments (sense orientation) were inserted between the chloramphenicol acetyl transferase (CAT) and luciferase (Luc) reporter genes. c) Translation efficiency of the mutant IRES elements in RRL. Equal amounts of in vitro synthesized RNAs pBIC-5, 101+1, 101Δ17 or 101Δ50 were used to program translation using Lb-untreated (-) RRL or Lb-treated (+) RRL. Proteins were resolved by SDS–PAGE and visualized by autoradiography of dry gels. Synthesis of luciferase driven by the indicated IRES elements in the presence or absence of the Lb protease is shown on the insert. The histogram shows the intensity of luciferase translation efficiency driven by the indicated RNAs in the presence of Lb (cap-independent).

    Techniques Used: Functional Assay, Sequencing, Construct, Plasmid Preparation, Positive Control, Chloramphenicol Acetyltransferase Assay, Luciferase, Mutagenesis, In Vitro, Synthesized, SDS Page, Autoradiography

    5) Product Images from "Microenvironment alters epigenetic and gene expression profiles in Swarm rat chondrosarcoma tumors"

    Article Title: Microenvironment alters epigenetic and gene expression profiles in Swarm rat chondrosarcoma tumors

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-10-471

    SAGE and quantitative RT-PCR confirm the expression of thymosin-β4 and c-fos in the SRC tumors . (A) Thymosin-β4 expression in the SRC tumors (SAGE analysis top panel; quantitative RT-PCR bottom panel). (B) C-fos expression in the SRC tumors (SAGE analysis top panel; quantitative RT-PCR bottom panel). SAGE and RT-PCR identified similar gene expression patterns for both thymosin-β4 and c-fos. Note the increased expression of thymosin-β4 and c-fos in the tibia and lung SRC tumors. The graphical bars in the RT-PCR figure represent the average expression ratio calculated on RNA that was collected from pooled tumor tissue. For RT-PCR at each transplantation site, tissue was pooled from at least 10 separate tumors. SAGE data was normalized to 100,000 tags/library for analysis. In the SAGE graph, 1.96). " title="... figure represent the average expression ratio calculated on RNA that was collected from pooled tumor tissue. For ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: SAGE and quantitative RT-PCR confirm the expression of thymosin-β4 and c-fos in the SRC tumors . (A) Thymosin-β4 expression in the SRC tumors (SAGE analysis top panel; quantitative RT-PCR bottom panel). (B) C-fos expression in the SRC tumors (SAGE analysis top panel; quantitative RT-PCR bottom panel). SAGE and RT-PCR identified similar gene expression patterns for both thymosin-β4 and c-fos. Note the increased expression of thymosin-β4 and c-fos in the tibia and lung SRC tumors. The graphical bars in the RT-PCR figure represent the average expression ratio calculated on RNA that was collected from pooled tumor tissue. For RT-PCR at each transplantation site, tissue was pooled from at least 10 separate tumors. SAGE data was normalized to 100,000 tags/library for analysis. In the SAGE graph, "*" indicates that the expression levels of a specific sample are significantly different relative to the "Subcutaneous SRC tumor" sample (z > 1.96).

    Techniques Used: Quantitative RT-PCR, Expressing, Reverse Transcription Polymerase Chain Reaction, Transplantation Assay

    6) Product Images from "Protein tyrosine phosphatase controls breast cancer invasion through the expression of matrix metalloproteinase-9"

    Article Title: Protein tyrosine phosphatase controls breast cancer invasion through the expression of matrix metalloproteinase-9

    Journal: BMB Reports

    doi: 10.5483/BMBRep.2013.46.11.053

    Effects of BVT948 on the viability of MCF-7 cells and TPA-induced MMP-9 expression. Cells were cultured in 96-well plates until 90% confluence, and various concentrations of BVT948 were then added to cells for 24 h. An established MTT assay was used to detect the viability of the cells (A). MCF-7 cells were treated with the indicated BVT948 concentrations in the presence of TPA for 24 h. MMP-9 mRNA levels were analyzed by real-time PCR, and GAPDH was used as an internal control (B). Cell lysates were analyzed by Western blot with an anti-MMP-9 antibody. The blot was retaken with anti β-actin to confirm equal loading (C). Conditioned medium was prepared and used for gelatin zymography (D). Each value represents the mean ± SEM of three independent experiments. *P < 0.01 vs. TPA.
    Figure Legend Snippet: Effects of BVT948 on the viability of MCF-7 cells and TPA-induced MMP-9 expression. Cells were cultured in 96-well plates until 90% confluence, and various concentrations of BVT948 were then added to cells for 24 h. An established MTT assay was used to detect the viability of the cells (A). MCF-7 cells were treated with the indicated BVT948 concentrations in the presence of TPA for 24 h. MMP-9 mRNA levels were analyzed by real-time PCR, and GAPDH was used as an internal control (B). Cell lysates were analyzed by Western blot with an anti-MMP-9 antibody. The blot was retaken with anti β-actin to confirm equal loading (C). Conditioned medium was prepared and used for gelatin zymography (D). Each value represents the mean ± SEM of three independent experiments. *P < 0.01 vs. TPA.

    Techniques Used: Expressing, Cell Culture, MTT Assay, Real-time Polymerase Chain Reaction, Western Blot, Zymography

    7) Product Images from "Recognition of Transmembrane Protein 39A as a Tumor-Specific Marker in Brain Tumor"

    Article Title: Recognition of Transmembrane Protein 39A as a Tumor-Specific Marker in Brain Tumor

    Journal: Toxicological Research

    doi: 10.5487/TR.2017.33.1.063

    Relative differences in TMEM39A transcript levels in GBM cells. Total RNAs were isolated from two GBM cell lines (U87-MG and U251-MG) and normal brain tissue. These samples were analyzed by standard RNA deep-sequencing (RNA-seq), as described in Materials and Methods. RNA-seq read densities of TMEM39A transcripts were plotted against relative RNA-seq read coverages (counts). “Fragments per kilobase of exon per million fragments mapped” (FPKMs) were calculated to compare the expression levels of TMEM39A mRNA variants among various sample.
    Figure Legend Snippet: Relative differences in TMEM39A transcript levels in GBM cells. Total RNAs were isolated from two GBM cell lines (U87-MG and U251-MG) and normal brain tissue. These samples were analyzed by standard RNA deep-sequencing (RNA-seq), as described in Materials and Methods. RNA-seq read densities of TMEM39A transcripts were plotted against relative RNA-seq read coverages (counts). “Fragments per kilobase of exon per million fragments mapped” (FPKMs) were calculated to compare the expression levels of TMEM39A mRNA variants among various sample.

    Techniques Used: Isolation, Sequencing, RNA Sequencing Assay, Expressing

    8) Product Images from "Translational Control of Mitochondrial Energy Production Mediates Neuron Morphogenesis"

    Article Title: Translational Control of Mitochondrial Energy Production Mediates Neuron Morphogenesis

    Journal: Cell metabolism

    doi: 10.1016/j.cmet.2012.11.002

    CPEB1 Regulates the Expression of Complex I Protein NDUFV2 (A) Blue native PAGE-resolved intact ETC complexes from mitochondria isolated from three WT and three CPEB1 KO brains and selected proteins in complexes I–V were analyzed by western blotting. Quantification is shown on the histogram. (B) Western blots were probed for mitochondrial proteins from total lysates from three WT and three CPEB1 KO brains. Quantification is shown on the histogram. (C) Quantitative RT-PCR analysis of NDUFV2, NDUFS4, and tubulin mRNAs in three WT and three CPEB1 KO brains. *p
    Figure Legend Snippet: CPEB1 Regulates the Expression of Complex I Protein NDUFV2 (A) Blue native PAGE-resolved intact ETC complexes from mitochondria isolated from three WT and three CPEB1 KO brains and selected proteins in complexes I–V were analyzed by western blotting. Quantification is shown on the histogram. (B) Western blots were probed for mitochondrial proteins from total lysates from three WT and three CPEB1 KO brains. Quantification is shown on the histogram. (C) Quantitative RT-PCR analysis of NDUFV2, NDUFS4, and tubulin mRNAs in three WT and three CPEB1 KO brains. *p

    Techniques Used: Expressing, Blue Native PAGE, Isolation, Western Blot, Quantitative RT-PCR

    CPEB1 Regulates NDUFV2 mRNA Translation (A) Schematic of NDUFV2 mRNA showing the mitochondrial targeting signal, polyadenylation hexanucleotide AAUAAA, and CPEs. (B) ATP was measured in WT and CPEB1 KO hippocampal neurons infected with lentiviruses expressing NDUFV2, or a mutant NDUFV2 lacking the MTS or NDUFS4. (C) Complex I activity was measured in WT and CPEB1 KO hippocampal neurons infected with lentivirus expressing NDUFV2. (D) WT hippocampal neurons were infected with lentivirus expressing HA-CPEB1. HA antibody was used to coimmunoprecipitate CPEB1 followed by RT-PCR for NDUFV2, NDUFS4, and GAPDH mRNAs. (E) WT and CPEB1 KO hippocampal neurons were incubated with 35 S-methionine followed by immunoprecipitation of NDUFV2 and analysis by SDS-PAGE. WCE refers to whole cell extract. The histogram shows the quantification of immunoprecipitation. (F) The coding region of Renilla luciferase was appended with the WT NDUFV2 3′UTR or one that lacked the CPEs (3′UTR mut) (see A). Plasmids encoding these constructs as well as firefly luciferase to serve as a control were transfected into neurons and analyzed for luciferase activity 3 days later. The data are plotted as the ratio of Renilla luciferase activity to firefly luciferase activity. (G) A PCR-based polyadenylation assay was used to determine the poly(A) tails of NDUFV2 and tubulin mRNAs in WT and CPEB1 KO brain (primers a/dT anchor). The internal primers (a/b) indicate the relative amount of RNA in each sample. *p
    Figure Legend Snippet: CPEB1 Regulates NDUFV2 mRNA Translation (A) Schematic of NDUFV2 mRNA showing the mitochondrial targeting signal, polyadenylation hexanucleotide AAUAAA, and CPEs. (B) ATP was measured in WT and CPEB1 KO hippocampal neurons infected with lentiviruses expressing NDUFV2, or a mutant NDUFV2 lacking the MTS or NDUFS4. (C) Complex I activity was measured in WT and CPEB1 KO hippocampal neurons infected with lentivirus expressing NDUFV2. (D) WT hippocampal neurons were infected with lentivirus expressing HA-CPEB1. HA antibody was used to coimmunoprecipitate CPEB1 followed by RT-PCR for NDUFV2, NDUFS4, and GAPDH mRNAs. (E) WT and CPEB1 KO hippocampal neurons were incubated with 35 S-methionine followed by immunoprecipitation of NDUFV2 and analysis by SDS-PAGE. WCE refers to whole cell extract. The histogram shows the quantification of immunoprecipitation. (F) The coding region of Renilla luciferase was appended with the WT NDUFV2 3′UTR or one that lacked the CPEs (3′UTR mut) (see A). Plasmids encoding these constructs as well as firefly luciferase to serve as a control were transfected into neurons and analyzed for luciferase activity 3 days later. The data are plotted as the ratio of Renilla luciferase activity to firefly luciferase activity. (G) A PCR-based polyadenylation assay was used to determine the poly(A) tails of NDUFV2 and tubulin mRNAs in WT and CPEB1 KO brain (primers a/dT anchor). The internal primers (a/b) indicate the relative amount of RNA in each sample. *p

    Techniques Used: Infection, Expressing, Mutagenesis, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Incubation, Immunoprecipitation, SDS Page, Luciferase, Construct, Transfection, Polymerase Chain Reaction

    9) Product Images from "Molecular Cloning Reveals that the p160 Myb-Binding Protein Is a Novel, Predominantly Nucleolar Protein Which May Play a Role in Transactivation by Myb"

    Article Title: Molecular Cloning Reveals that the p160 Myb-Binding Protein Is a Novel, Predominantly Nucleolar Protein Which May Play a Role in Transactivation by Myb

    Journal: Molecular and Cellular Biology

    doi:

    Northern blot analysis of p160 mRNA expression with the p160 cDNA probe. In the upper panels, 2 μg of poly(A) + RNA from the indicated cell lines was loaded in each lane (MTHC, myb transformed hemopoietic cells) (A) or 30 μg of total RNA from the indicated mouse tissues (or FDC-P1 cells) was loaded in each lane (B). The size of the p160 mRNA was determined by comparison with RNA markers (not shown). In the lower panels, the Northern blots shown in the upper panels were stripped and reprobed with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA as a control for mRNA loading; only the relevant area of each blot is shown.
    Figure Legend Snippet: Northern blot analysis of p160 mRNA expression with the p160 cDNA probe. In the upper panels, 2 μg of poly(A) + RNA from the indicated cell lines was loaded in each lane (MTHC, myb transformed hemopoietic cells) (A) or 30 μg of total RNA from the indicated mouse tissues (or FDC-P1 cells) was loaded in each lane (B). The size of the p160 mRNA was determined by comparison with RNA markers (not shown). In the lower panels, the Northern blots shown in the upper panels were stripped and reprobed with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA as a control for mRNA loading; only the relevant area of each blot is shown.

    Techniques Used: Northern Blot, Expressing, Transformation Assay

    10) Product Images from "A Common Genetic Variant (97906C > A) of DAB2IP/AIP1 Is Associated with an Increased Risk and Early Onset of Lung Cancer in Chinese Males"

    Article Title: A Common Genetic Variant (97906C > A) of DAB2IP/AIP1 Is Associated with an Increased Risk and Early Onset of Lung Cancer in Chinese Males

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0026944

    Association of the 97906C > A genotype and DAB2IP expressions. A, Relative mRNA levels of the DAB2IP expressions in lung cancer tissues compared to their adjacent normal lung tissues; B, Relative mRNA level of the DAB2IP expression by the 97906C > A genotype in lung cancer tissues. C, Relative mRNA level of the DAB2IP expression by the 97906C > A genotype in normal tissues. No significant association was observed between the 97906C > A genotype and DAB2IP mRNA levels neither in cancer tissues nor in normal tissues Columns, mean from three independent experiments; bars, SD; and Student's t test was used to test the differences in the expression levels of different constructs.
    Figure Legend Snippet: Association of the 97906C > A genotype and DAB2IP expressions. A, Relative mRNA levels of the DAB2IP expressions in lung cancer tissues compared to their adjacent normal lung tissues; B, Relative mRNA level of the DAB2IP expression by the 97906C > A genotype in lung cancer tissues. C, Relative mRNA level of the DAB2IP expression by the 97906C > A genotype in normal tissues. No significant association was observed between the 97906C > A genotype and DAB2IP mRNA levels neither in cancer tissues nor in normal tissues Columns, mean from three independent experiments; bars, SD; and Student's t test was used to test the differences in the expression levels of different constructs.

    Techniques Used: Expressing, Construct

    11) Product Images from "Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing"

    Article Title: Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr693

    Comparison of rankings between the standard adapters (noNN,ranks along x -axis) versus fNN_eNN (ranks along y -axis) for 293T ( A ) and mES samples ( B ). A point above the diagonal represents a sequence that is overrepresented in noNN, while below the diagonal are points that are underrepresented in noNN. The hsa-miR-18a is overrepresented in the noNN case, where it is ranked 3, the array and qPCR data agree better with the fNN_eNN results which ranks it much lower [this skew is also seen in the mES samples, but the ranking in the noNN is 22 while the fNN_eNN is much lower (135)]. In the mES sample, mmu-miR-294 is first and a non-canonical form of mmu-mir-292-3p is second for noNN, while they switch ranks in the fNN_eNN case, the difference is very significant, because the abundances of the first and the second ranks are about 2-fold apart, suggesting a strong bias. mmu-miR-290-5p is very high at rank 5 in the case of noNN, it is outside the range of the graph in fNN_eNN, in accordance with the qPCR data. Thus, in every case that we can detect a difference between noNN and fNN_eNN, fNN_eNN seems to be more accurate in reflecting the profiles.
    Figure Legend Snippet: Comparison of rankings between the standard adapters (noNN,ranks along x -axis) versus fNN_eNN (ranks along y -axis) for 293T ( A ) and mES samples ( B ). A point above the diagonal represents a sequence that is overrepresented in noNN, while below the diagonal are points that are underrepresented in noNN. The hsa-miR-18a is overrepresented in the noNN case, where it is ranked 3, the array and qPCR data agree better with the fNN_eNN results which ranks it much lower [this skew is also seen in the mES samples, but the ranking in the noNN is 22 while the fNN_eNN is much lower (135)]. In the mES sample, mmu-miR-294 is first and a non-canonical form of mmu-mir-292-3p is second for noNN, while they switch ranks in the fNN_eNN case, the difference is very significant, because the abundances of the first and the second ranks are about 2-fold apart, suggesting a strong bias. mmu-miR-290-5p is very high at rank 5 in the case of noNN, it is outside the range of the graph in fNN_eNN, in accordance with the qPCR data. Thus, in every case that we can detect a difference between noNN and fNN_eNN, fNN_eNN seems to be more accurate in reflecting the profiles.

    Techniques Used: Sequencing, Real-time Polymerase Chain Reaction

    The two terminal 3′ bases of the 5′ adapter are the primary determinants of T4-RNA ligase 1 (Rnl1) ligation efficiency. Two distinct sets of 5′ adapters, one consisting of adapters with mixed bases in the last four bases (fNNNN) and another consisting of adapters with mixed bases in the last two bases (fNN), were used to generate a miRNA derived cDNA library for ( A ) human 293T and ( B ) mouse embryonic stem cell lines. miRNA abundance in read counts (dots) were plotted; the fNNNN data were compressed to NN, by combining values for AANN through TTNN for each NN. The high correlation between the compressed fNNNN and the fNN datasets indicates that the two terminal bases are dominant determinants of ligation efficiency. There are exceptions shown in red, which are systematic differences (106b, 181 in 293T cell), which we detected in an independent experiment described in Figure 2 suggesting that this is not a stochastic effect. The naming convention in all our figures is to show the beginning and end of the sequence followed by an m (for a canonical mature) or n for a non-canonical miRNA sequence followed by the name of the miRNA. Thus in the left we have a canonical mature hsa-miR-106-b and a non-canonical hsa-miR-218. The high abundance for hsa-miR-106b suggested by the fNNNN strategy (in contrast to the low values suggested by fNN and other strategies) seems real, as the microarray and RT–PCR results ( Figure 9 ) are in concordance with the fNNNN values.
    Figure Legend Snippet: The two terminal 3′ bases of the 5′ adapter are the primary determinants of T4-RNA ligase 1 (Rnl1) ligation efficiency. Two distinct sets of 5′ adapters, one consisting of adapters with mixed bases in the last four bases (fNNNN) and another consisting of adapters with mixed bases in the last two bases (fNN), were used to generate a miRNA derived cDNA library for ( A ) human 293T and ( B ) mouse embryonic stem cell lines. miRNA abundance in read counts (dots) were plotted; the fNNNN data were compressed to NN, by combining values for AANN through TTNN for each NN. The high correlation between the compressed fNNNN and the fNN datasets indicates that the two terminal bases are dominant determinants of ligation efficiency. There are exceptions shown in red, which are systematic differences (106b, 181 in 293T cell), which we detected in an independent experiment described in Figure 2 suggesting that this is not a stochastic effect. The naming convention in all our figures is to show the beginning and end of the sequence followed by an m (for a canonical mature) or n for a non-canonical miRNA sequence followed by the name of the miRNA. Thus in the left we have a canonical mature hsa-miR-106-b and a non-canonical hsa-miR-218. The high abundance for hsa-miR-106b suggested by the fNNNN strategy (in contrast to the low values suggested by fNN and other strategies) seems real, as the microarray and RT–PCR results ( Figure 9 ) are in concordance with the fNNNN values.

    Techniques Used: Ligation, Derivative Assay, cDNA Library Assay, Sequencing, Microarray, Reverse Transcription Polymerase Chain Reaction

    Comparison of sequencing against microarray ( A and B ) and RT–PCR ( C and D ) for mES (B and D) and 293T (A and C). There are outliers, such as miR-106b, which are only captured by the fNNNN strategy, but overall, there is significant correlation between the fNN_eNN strategy and the microarray data (A) and the fNN_eNN strategy and the RT–PCR data (C), while the fNN sequencing strategy does not give a good correlation to RT–PCR and array data (B and D).
    Figure Legend Snippet: Comparison of sequencing against microarray ( A and B ) and RT–PCR ( C and D ) for mES (B and D) and 293T (A and C). There are outliers, such as miR-106b, which are only captured by the fNNNN strategy, but overall, there is significant correlation between the fNN_eNN strategy and the microarray data (A) and the fNN_eNN strategy and the RT–PCR data (C), while the fNN sequencing strategy does not give a good correlation to RT–PCR and array data (B and D).

    Techniques Used: Sequencing, Microarray, Reverse Transcription Polymerase Chain Reaction

    Fluctuation plots showing ligation efficiency for different fNN (A and C) and eNN (B and D) adapters against the most abundant miRNAs from 293T ( A and B ) and mES ( C and D ) cells. The naming convention in all our figures is to show the beginning and end of the sequence followed by an m (for a canonical mature) or n for a non-canonical miRNA sequence followed by the name of the miRNA. The area of the dark rectangles depicts the value for each combination of miRNA and adapter. The standard adapter ends (TC in fNN and CT in eNN, highlighted in gray boxes) are not very efficient in ligation to the most abundant miRNAs. Even the most efficient adapters show variability, suggesting that no single adapter can work well across all possible sequences. For the top miRNAs, most of the variability comes from the 3′ adapter ligation (the eNN adapters, B and D). In mES cells, there are two isomirs of mmu-miR-292-3p, the GT ending 3′ adapter captures the GAGT-ending isomir more efficiently, while the GA ending 3′ adapter captures the GAGTG-ending isomir more efficiently.
    Figure Legend Snippet: Fluctuation plots showing ligation efficiency for different fNN (A and C) and eNN (B and D) adapters against the most abundant miRNAs from 293T ( A and B ) and mES ( C and D ) cells. The naming convention in all our figures is to show the beginning and end of the sequence followed by an m (for a canonical mature) or n for a non-canonical miRNA sequence followed by the name of the miRNA. The area of the dark rectangles depicts the value for each combination of miRNA and adapter. The standard adapter ends (TC in fNN and CT in eNN, highlighted in gray boxes) are not very efficient in ligation to the most abundant miRNAs. Even the most efficient adapters show variability, suggesting that no single adapter can work well across all possible sequences. For the top miRNAs, most of the variability comes from the 3′ adapter ligation (the eNN adapters, B and D). In mES cells, there are two isomirs of mmu-miR-292-3p, the GT ending 3′ adapter captures the GAGT-ending isomir more efficiently, while the GA ending 3′ adapter captures the GAGTG-ending isomir more efficiently.

    Techniques Used: Ligation, Sequencing

    A radar plot showing the performance of different adapter termini combinations (fNN_eNN), shown outside the circle in blue. The inner circles represent percent contribution of each adapter combination to a particular miRNA that was sequenced. This plot shows data for the top miRNA (hsa-miR-20a) in 293T cells and two top miRNAs (mmu-miR-292-3p and mmu-miR-294) from mouse embryonic stem cells. There is large variation in the efficiency of capture between various combinations of 5′ and 3′ adapter end modifications. This emphasizes the need for a pooled strategy in sequencing.
    Figure Legend Snippet: A radar plot showing the performance of different adapter termini combinations (fNN_eNN), shown outside the circle in blue. The inner circles represent percent contribution of each adapter combination to a particular miRNA that was sequenced. This plot shows data for the top miRNA (hsa-miR-20a) in 293T cells and two top miRNAs (mmu-miR-292-3p and mmu-miR-294) from mouse embryonic stem cells. There is large variation in the efficiency of capture between various combinations of 5′ and 3′ adapter end modifications. This emphasizes the need for a pooled strategy in sequencing.

    Techniques Used: Sequencing

    12) Product Images from "The Role of IL-17 Promotes Spinal Cord Neuroinflammation via Activation of the Transcription Factor STAT3 after Spinal Cord Injury in the Rat"

    Article Title: The Role of IL-17 Promotes Spinal Cord Neuroinflammation via Activation of the Transcription Factor STAT3 after Spinal Cord Injury in the Rat

    Journal: Mediators of Inflammation

    doi: 10.1155/2014/786947

    (a) and (b): IL-17 and IL-23p19 mRNA transcription in spleen tissue. Representative images showed semiquantitative RT-PCR for IL-17 and IL-23p19 transcription sham. The sham-operated group; 1 h. The 1 h group of SCI; 24 h. The 24 h group of SCI; 48 h. The 48 h group of SCI; 72 h. The 72 h group of SCI. (c) and (d): densitometric quantification of IL-17 and IL-23p19mRNA expression in spleen tissues. Densitometric quantification of PCR bands showed that, among the IL-17 and IL-23p19, the sham-operated group, 1 h group of SCI, 24 h group of SCI, 48 h group of SCI, and 72 h group of SCI, the mRNA expression was significantly difference between each groups and peaked at 24 h. The data are presented as the mean ± the SE. * P
    Figure Legend Snippet: (a) and (b): IL-17 and IL-23p19 mRNA transcription in spleen tissue. Representative images showed semiquantitative RT-PCR for IL-17 and IL-23p19 transcription sham. The sham-operated group; 1 h. The 1 h group of SCI; 24 h. The 24 h group of SCI; 48 h. The 48 h group of SCI; 72 h. The 72 h group of SCI. (c) and (d): densitometric quantification of IL-17 and IL-23p19mRNA expression in spleen tissues. Densitometric quantification of PCR bands showed that, among the IL-17 and IL-23p19, the sham-operated group, 1 h group of SCI, 24 h group of SCI, 48 h group of SCI, and 72 h group of SCI, the mRNA expression was significantly difference between each groups and peaked at 24 h. The data are presented as the mean ± the SE. * P

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

    13) Product Images from "Depletion of the RNA-Binding Protein RBP33 Results in Increased Expression of Silenced RNA Polymerase II Transcripts in Trypanosoma brucei"

    Article Title: Depletion of the RNA-Binding Protein RBP33 Results in Increased Expression of Silenced RNA Polymerase II Transcripts in Trypanosoma brucei

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0107608

    RBP33 is an essential protein in trypanosomes. Cell lines were generated that expressed RBP33 -specific dsRNA in a tetracycline-inducible manner. The levels of mRNA (A) or protein (B) were analyzed in cells incubated with tetracycline for 48 h. Loading controls were the 7SL RNA [40] or the CSM protein [39] . (C) RNAi cell lines were grown in the absence (open squares) or presence (filled circles) of 1 µg/ml tetracycline to induce RNAi. Cultures were followed up to 8 days and diluted to 0.5×10 5 cells/ml (bloodstream) or 0.4×10 6 cells/ml (procyclic) every 2 days as required.
    Figure Legend Snippet: RBP33 is an essential protein in trypanosomes. Cell lines were generated that expressed RBP33 -specific dsRNA in a tetracycline-inducible manner. The levels of mRNA (A) or protein (B) were analyzed in cells incubated with tetracycline for 48 h. Loading controls were the 7SL RNA [40] or the CSM protein [39] . (C) RNAi cell lines were grown in the absence (open squares) or presence (filled circles) of 1 µg/ml tetracycline to induce RNAi. Cultures were followed up to 8 days and diluted to 0.5×10 5 cells/ml (bloodstream) or 0.4×10 6 cells/ml (procyclic) every 2 days as required.

    Techniques Used: Generated, Incubation

    Expression and subcellular localization of RBP33 in bloodstream and procyclic trypanosomes. (A) Schematic diagram of T. brucei RBP33. An anti-RBP33 antiserum was used to detect the protein by (B) western blot analysis of total cell extracts or (C) immunofluorescence assays. CSM protein [39] was included as loading control, and DAPI was used to stain nuclei.
    Figure Legend Snippet: Expression and subcellular localization of RBP33 in bloodstream and procyclic trypanosomes. (A) Schematic diagram of T. brucei RBP33. An anti-RBP33 antiserum was used to detect the protein by (B) western blot analysis of total cell extracts or (C) immunofluorescence assays. CSM protein [39] was included as loading control, and DAPI was used to stain nuclei.

    Techniques Used: Expressing, Western Blot, Immunofluorescence, Staining

    Distribution of RBP33-bound transcripts. (A) RNAs associated with RBP33 (log 2 FC > 3) were categorized according to whether they are predicted to encode functional proteins. Color code is based on that of GeneDB ( http://www.genedb.org/Homepage/Tbruceibrucei927 ). (B) Localization of the genes encoding RBP33 targets (log 2 FC > 3) on the genome. ‘SSR’ refers to genes localized in the vicinity of strand-switch regions, whereas ‘Other’ indicates genes found close to the beginning or end of chromosomes, or close to snoRNA or pseudogene clusters.
    Figure Legend Snippet: Distribution of RBP33-bound transcripts. (A) RNAs associated with RBP33 (log 2 FC > 3) were categorized according to whether they are predicted to encode functional proteins. Color code is based on that of GeneDB ( http://www.genedb.org/Homepage/Tbruceibrucei927 ). (B) Localization of the genes encoding RBP33 targets (log 2 FC > 3) on the genome. ‘SSR’ refers to genes localized in the vicinity of strand-switch regions, whereas ‘Other’ indicates genes found close to the beginning or end of chromosomes, or close to snoRNA or pseudogene clusters.

    Techniques Used: Functional Assay

    Effect of RBP33 depletion on the levels of SSR-derived transcripts. (A) Artmemis visualization of RNAseq data corresponding to the SSR downstream gene Tb927.7.1930 and to the SSR downstream gene Tb927.10.5710. Probes used for Northern blot analysis are indicated. ‘Input’ refers to the RNA-seq data obtained from total RNA before immunoprecipitation, whereas ‘RBP33-bound’ refers to the immunoprecipitated RNA using RBP33 specific antibodies. Annotated splice-acceptor and polyadenylation sites [13] are indicated as filled or empty circles, respectively. The splice-acceptor site for Tb927.10.5720 has been mapped in this study, and lies within the downstream open-reading frame. (B) Northern hybridizations using radiolabeled double-stranded DNA probes as indicated in panel A. An RNAi cell line expressing dsRNA against the essential RNA-binding protein DRBD3 [11] was used as a control. Total RNA was obtained from parasites grown in the absence (−) or the presence (+) of tetracycline for 48 h, transferred to nylon membranes and hybridized with specific probes. The 7SL RNA was used as a loading control. The arrow indicates the position of the 1.0 kb transcript detected with probe B. Representative blots are shown.
    Figure Legend Snippet: Effect of RBP33 depletion on the levels of SSR-derived transcripts. (A) Artmemis visualization of RNAseq data corresponding to the SSR downstream gene Tb927.7.1930 and to the SSR downstream gene Tb927.10.5710. Probes used for Northern blot analysis are indicated. ‘Input’ refers to the RNA-seq data obtained from total RNA before immunoprecipitation, whereas ‘RBP33-bound’ refers to the immunoprecipitated RNA using RBP33 specific antibodies. Annotated splice-acceptor and polyadenylation sites [13] are indicated as filled or empty circles, respectively. The splice-acceptor site for Tb927.10.5720 has been mapped in this study, and lies within the downstream open-reading frame. (B) Northern hybridizations using radiolabeled double-stranded DNA probes as indicated in panel A. An RNAi cell line expressing dsRNA against the essential RNA-binding protein DRBD3 [11] was used as a control. Total RNA was obtained from parasites grown in the absence (−) or the presence (+) of tetracycline for 48 h, transferred to nylon membranes and hybridized with specific probes. The 7SL RNA was used as a loading control. The arrow indicates the position of the 1.0 kb transcript detected with probe B. Representative blots are shown.

    Techniques Used: Derivative Assay, Northern Blot, RNA Sequencing Assay, Immunoprecipitation, Expressing, RNA Binding Assay

    Validation of RBP33 targets. Quantitative RT-PCR assays were performed to confirm binding of three coding RNAs (pteridine transporter PT-X , Tb927.10.9080; flagellar transition zone component FTZC , Tb927.10.8590; and Tb927.10.4720) and transcripts derived from the SSR downstream gene Tb927.10.5720 or from the SSR downstream gene Tb927.2.3320.
    Figure Legend Snippet: Validation of RBP33 targets. Quantitative RT-PCR assays were performed to confirm binding of three coding RNAs (pteridine transporter PT-X , Tb927.10.9080; flagellar transition zone component FTZC , Tb927.10.8590; and Tb927.10.4720) and transcripts derived from the SSR downstream gene Tb927.10.5720 or from the SSR downstream gene Tb927.2.3320.

    Techniques Used: Quantitative RT-PCR, Binding Assay, Derivative Assay

    Effect of RBP33 depletion on the levels of transcripts derived from retroposons and CIR147 repeats. Probes designed to detect both INGI/RIME-derived transcripts, SLACs retroposons or CIR147-derived transcripts were used in Northern hybridizations as described in Fig. 5 legend. Ethidium bromide staining was used as a loading control. Vertical lines indicate the expected migration of the corresponding transcripts upon disruption of the RNAi machinery observed in [6] .
    Figure Legend Snippet: Effect of RBP33 depletion on the levels of transcripts derived from retroposons and CIR147 repeats. Probes designed to detect both INGI/RIME-derived transcripts, SLACs retroposons or CIR147-derived transcripts were used in Northern hybridizations as described in Fig. 5 legend. Ethidium bromide staining was used as a loading control. Vertical lines indicate the expected migration of the corresponding transcripts upon disruption of the RNAi machinery observed in [6] .

    Techniques Used: Derivative Assay, Northern Blot, Staining, Migration

    14) Product Images from "STAT1-activating cytokines limit Th17 responses through both T-bet-dependent and independent mechanisms 1"

    Article Title: STAT1-activating cytokines limit Th17 responses through both T-bet-dependent and independent mechanisms 1

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1001343

    Kinetics of Th1 and Th17 responses during systemic autoimmune disease (A) Naïve DO11.10 CD4 + T cells were transferred into Rag2−/− or sOva Rag2−/− hosts. 3–9 days later, lymphocytes from recipient mice were re-stimulated ex vivo and cytokine production measured by intracellular flow cytometry. Shown is the percentage of cytokine + donor cells (CD4 + DO11.10 + ) at the indicated time points. Day 0 represents cytokine production in naïve controls. Data are compiled from 5 experiments (5–10 mice/group) and shown is the fold change for sOva Rag2−/− hosts comparing the early and late time points. (B) Adoptive transfers were performed as in (A). CD4 + DO11.10 + CD44 high donor cells were purified by high-speed cell sorting and mRNA levels quantified by real-time PCR. Data are representative of 4 experiments and are presented as the fold increase (X > 1) or decrease (X
    Figure Legend Snippet: Kinetics of Th1 and Th17 responses during systemic autoimmune disease (A) Naïve DO11.10 CD4 + T cells were transferred into Rag2−/− or sOva Rag2−/− hosts. 3–9 days later, lymphocytes from recipient mice were re-stimulated ex vivo and cytokine production measured by intracellular flow cytometry. Shown is the percentage of cytokine + donor cells (CD4 + DO11.10 + ) at the indicated time points. Day 0 represents cytokine production in naïve controls. Data are compiled from 5 experiments (5–10 mice/group) and shown is the fold change for sOva Rag2−/− hosts comparing the early and late time points. (B) Adoptive transfers were performed as in (A). CD4 + DO11.10 + CD44 high donor cells were purified by high-speed cell sorting and mRNA levels quantified by real-time PCR. Data are representative of 4 experiments and are presented as the fold increase (X > 1) or decrease (X

    Techniques Used: Mouse Assay, Ex Vivo, Flow Cytometry, Cytometry, Purification, FACS, Real-time Polymerase Chain Reaction

    15) Product Images from "Distinct chromatin functional states correlate with HIV latency reactivation in infected primary CD4+ T cells"

    Article Title: Distinct chromatin functional states correlate with HIV latency reactivation in infected primary CD4+ T cells

    Journal: eLife

    doi: 10.7554/eLife.34655

    LRAs efficacy in patient samples is predicted by activity in HIV GKO latently infected cells. ( A ). ( B ) Intracellular HIV-1 mRNA levels in HIV GKO latently infected CD4 + ). ( C ) Correlation between intracellular HIV-1 mRNA levels quantified in either 6 hr stimulated HIV GKO latently infected CD4 + T-cells from different donors, or 24 hr stimulated rCD4s from HIV infected patients, with a single LRA or a combination of two LRAs in presence of raltegravir.
    Figure Legend Snippet: LRAs efficacy in patient samples is predicted by activity in HIV GKO latently infected cells. ( A ). ( B ) Intracellular HIV-1 mRNA levels in HIV GKO latently infected CD4 + ). ( C ) Correlation between intracellular HIV-1 mRNA levels quantified in either 6 hr stimulated HIV GKO latently infected CD4 + T-cells from different donors, or 24 hr stimulated rCD4s from HIV infected patients, with a single LRA or a combination of two LRAs in presence of raltegravir.

    Techniques Used: Activity Assay, Infection

    (1)Intracellular HIV-1 mRNA levels in rCD4s, obtained from infected individuals and treated ex vivo with a single LRA or a combination of two LRAs for 24 hours, presented as fold induction relative to DMSO control. (n = 4, mean + SEM).
    Figure Legend Snippet: (1)Intracellular HIV-1 mRNA levels in rCD4s, obtained from infected individuals and treated ex vivo with a single LRA or a combination of two LRAs for 24 hours, presented as fold induction relative to DMSO control. (n = 4, mean + SEM).

    Techniques Used: Infection, Ex Vivo

    16) Product Images from "STRL33, A Novel Chemokine Receptor-like Protein, Functions as a Fusion Cofactor for Both Macrophage-tropic and T Cell Line-tropic HIV-1"

    Article Title: STRL33, A Novel Chemokine Receptor-like Protein, Functions as a Fusion Cofactor for Both Macrophage-tropic and T Cell Line-tropic HIV-1

    Journal: The Journal of Experimental Medicine

    doi:

    Expression of STRL33 and other GPCR genes. ( A ) The expression of STRL33 , genes for known chemokine receptors, and genes for selected orphan GPCRs in leukocytes. 15 μg of total RNA were electrophoresed on 1.2% agarose–formaldehyde gels, transferred to nitrocellulose membranes, and hybridized with the probes indicated on the left. A total of six membranes were used for hybridizations, and adequate removal of signal was documented before repeat probings. Film exposure times ranged from overnight for the IL-8RA and IL-8RB blots to 13 d for the CXCR4 blot. Probings were done using an oligonucleotide complementary to 18S rRNA in order to demonstrate amounts of RNA loaded per lane and a representative blot is shown. ( B ) The expression of STRL33 in activated PBL. 25 μg of total RNA from TIL, and freshly isolated and activated PBL were analyzed as in A and hybridized with a 32 P-labeled STRL33 ORF probe and a probe for 18S rRNA.
    Figure Legend Snippet: Expression of STRL33 and other GPCR genes. ( A ) The expression of STRL33 , genes for known chemokine receptors, and genes for selected orphan GPCRs in leukocytes. 15 μg of total RNA were electrophoresed on 1.2% agarose–formaldehyde gels, transferred to nitrocellulose membranes, and hybridized with the probes indicated on the left. A total of six membranes were used for hybridizations, and adequate removal of signal was documented before repeat probings. Film exposure times ranged from overnight for the IL-8RA and IL-8RB blots to 13 d for the CXCR4 blot. Probings were done using an oligonucleotide complementary to 18S rRNA in order to demonstrate amounts of RNA loaded per lane and a representative blot is shown. ( B ) The expression of STRL33 in activated PBL. 25 μg of total RNA from TIL, and freshly isolated and activated PBL were analyzed as in A and hybridized with a 32 P-labeled STRL33 ORF probe and a probe for 18S rRNA.

    Techniques Used: Expressing, Isolation, Labeling

    17) Product Images from "Induced ectopic expression of HigB toxin in Mycobacterium tuberculosis results in growth inhibition, reduced abundance of a subset of mRNAs and cleavage of tmRNA"

    Article Title: Induced ectopic expression of HigB toxin in Mycobacterium tuberculosis results in growth inhibition, reduced abundance of a subset of mRNAs and cleavage of tmRNA

    Journal: Molecular Microbiology

    doi: 10.1111/mmi.12358

    HigB expression affects tmRNA in M. tuberculosis ΔTAC. RNA was extracted from mid-exponential cultures treated with ATc for 2 h, 6 h or 24 h and cDNA was prepared for qRT-PCR analysis.A–C. Northern blots probing for tmRNA (A and B) and 5S (C). Transcript sizes are relative to the position of RNA marker and tmRNA cleavage products are indicated with an arrow.D. Quantitative RT-PCR of transcripts of interest. All results are the mean values and standard deviation of three independent biological replicates. A significant difference (as determined by Student's t -test) between vector control and HigB overexpression strain is marked by an asterisk (*) for P
    Figure Legend Snippet: HigB expression affects tmRNA in M. tuberculosis ΔTAC. RNA was extracted from mid-exponential cultures treated with ATc for 2 h, 6 h or 24 h and cDNA was prepared for qRT-PCR analysis.A–C. Northern blots probing for tmRNA (A and B) and 5S (C). Transcript sizes are relative to the position of RNA marker and tmRNA cleavage products are indicated with an arrow.D. Quantitative RT-PCR of transcripts of interest. All results are the mean values and standard deviation of three independent biological replicates. A significant difference (as determined by Student's t -test) between vector control and HigB overexpression strain is marked by an asterisk (*) for P

    Techniques Used: Expressing, Quantitative RT-PCR, Northern Blot, Marker, Standard Deviation, Plasmid Preparation, Over Expression

    18) Product Images from "A parallel genome-wide mRNA and microRNA profiling of the frontal cortex of HIV patients with and without HIV-associated dementia shows the role of axon guidance and downstream pathways in HIV-mediated neurodegeneration"

    Article Title: A parallel genome-wide mRNA and microRNA profiling of the frontal cortex of HIV patients with and without HIV-associated dementia shows the role of axon guidance and downstream pathways in HIV-mediated neurodegeneration

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-13-677

    Validation of mRNA and miRNA profiles using quantitative RT-PCR on the same sample set of microarray study. A . mRNA profiles validation. B . miRNA profiles validation. Data was analysed using the 2 -ΔΔCt method and results were plotted as fold differences of relative expression normalized to house keeping gene (GAPDH) and Hs_RNU6B_3 individually. The data have been collected from the same RNA samples from which mRNA and miRNA profiling have been done. All the data presented in this figure have significant P value of
    Figure Legend Snippet: Validation of mRNA and miRNA profiles using quantitative RT-PCR on the same sample set of microarray study. A . mRNA profiles validation. B . miRNA profiles validation. Data was analysed using the 2 -ΔΔCt method and results were plotted as fold differences of relative expression normalized to house keeping gene (GAPDH) and Hs_RNU6B_3 individually. The data have been collected from the same RNA samples from which mRNA and miRNA profiling have been done. All the data presented in this figure have significant P value of

    Techniques Used: Quantitative RT-PCR, Microarray, Expressing

    19) Product Images from "Beneficial Effects of Myo-Inositol Oxygenase Deficiency in Cisplatin-Induced AKI"

    Article Title: Beneficial Effects of Myo-Inositol Oxygenase Deficiency in Cisplatin-Induced AKI

    Journal: Journal of the American Society of Nephrology : JASN

    doi: 10.1681/ASN.2016070744

    MIOX gene disruption inhibits, whereas its overexpression accentuates, renal tubular cell apoptosis. Expression of proapoptogenic Bax was increased after cisplatin treatment in kidney tubules of WT mice (D versus A, and M). It was highly accentuated in MIOX-TG mice (E versus B, and M). No increase was observed in MIOX-KO mice (F versus C, and M). Intriguingly, at times a mild decrease in BAX expression was noted in KO mice (M), whereas antiapoptogenic Bcl-2 expression drastically decreased in both the WT and MIOX-TG mice, and it was unchanged in KO mice (M). An increased expression of cleaved caspase-3 was observed in kidneys of WT mice after cisplatin treatment (M), which was accentuated in MIOX-TG mice. No expression of cleaved caspase-3 was observed in treated or untreated MIOX-KO mice (M). After cisplatin treatment, a marked increase in caspase-3 activity was observed in MIOX-TG mice, whereas a moderate increase was also observed in WT mice (N). No significant increase in caspase-3 was observed in kidneys of KO mice. Along these lines, a fulminant degree of apoptosis was observed in kidneys of MIOX-TG mice (K versus H), although a mild-to-moderate degree of apoptosis was also observed in WT mice (J versus G), and no apoptosis was detected in MIOX-KO mice (I and L).
    Figure Legend Snippet: MIOX gene disruption inhibits, whereas its overexpression accentuates, renal tubular cell apoptosis. Expression of proapoptogenic Bax was increased after cisplatin treatment in kidney tubules of WT mice (D versus A, and M). It was highly accentuated in MIOX-TG mice (E versus B, and M). No increase was observed in MIOX-KO mice (F versus C, and M). Intriguingly, at times a mild decrease in BAX expression was noted in KO mice (M), whereas antiapoptogenic Bcl-2 expression drastically decreased in both the WT and MIOX-TG mice, and it was unchanged in KO mice (M). An increased expression of cleaved caspase-3 was observed in kidneys of WT mice after cisplatin treatment (M), which was accentuated in MIOX-TG mice. No expression of cleaved caspase-3 was observed in treated or untreated MIOX-KO mice (M). After cisplatin treatment, a marked increase in caspase-3 activity was observed in MIOX-TG mice, whereas a moderate increase was also observed in WT mice (N). No significant increase in caspase-3 was observed in kidneys of KO mice. Along these lines, a fulminant degree of apoptosis was observed in kidneys of MIOX-TG mice (K versus H), although a mild-to-moderate degree of apoptosis was also observed in WT mice (J versus G), and no apoptosis was detected in MIOX-KO mice (I and L).

    Techniques Used: Over Expression, Expressing, Mouse Assay, Activity Assay

    20) Product Images from "The Herpesvirus saimiri Small Nuclear RNAs Recruit AU-Rich Element-Binding Proteins but Do Not Alter Host AU-Rich Element-Containing mRNA Levels in Virally Transformed T Cells"

    Article Title: The Herpesvirus saimiri Small Nuclear RNAs Recruit AU-Rich Element-Binding Proteins but Do Not Alter Host AU-Rich Element-Containing mRNA Levels in Virally Transformed T Cells

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.24.10.4522-4533.2004

    Northern analyses of host class I and class II ARE-containing mRNAs. Polyadenylated RNAs isolated from wild-type (Wt) and mutant (Mt) HVS-transformed T-cell lines were resolved in a 1% formaldehyde agarose gel, and Northern blotting was performed for the class I ARE-containing mRNAs c- myc (lanes 1 and 2) and IL-10 (lanes 3 and 4) and for the class II ARE-containing mRNAs TNF-α (lanes 5 and 6) and Pim-1 (lanes 7 and 8). The same membranes were probed for β-actin as a loading control. The membrane used for TNF-α was stripped and reprobed for Pim-1.
    Figure Legend Snippet: Northern analyses of host class I and class II ARE-containing mRNAs. Polyadenylated RNAs isolated from wild-type (Wt) and mutant (Mt) HVS-transformed T-cell lines were resolved in a 1% formaldehyde agarose gel, and Northern blotting was performed for the class I ARE-containing mRNAs c- myc (lanes 1 and 2) and IL-10 (lanes 3 and 4) and for the class II ARE-containing mRNAs TNF-α (lanes 5 and 6) and Pim-1 (lanes 7 and 8). The same membranes were probed for β-actin as a loading control. The membrane used for TNF-α was stripped and reprobed for Pim-1.

    Techniques Used: Northern Blot, Isolation, Mutagenesis, Transformation Assay, Agarose Gel Electrophoresis

    21) Product Images from "Vitamin B1 diversity and characterization of biosynthesis genes in cassava"

    Article Title: Vitamin B1 diversity and characterization of biosynthesis genes in cassava

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/erx196

    Organization of the cassava MeTHIC1 and MeTHIC2 3ʹ-UTR regions. PCR amplification of (A) MeTHIC1 and (B) MeTHIC2 3ʹ-UTR regions. Plants were grown in vitro in cassava basic medium without vitamin B 1 (indicated by 0) or in medium supplemented with 10 μM of vitamin B 1 (indicated by +B 1 ). cDNA was synthesized using oligo-(dT) 18 primers, random hexamer oligonucleotides, or oligo-(dT) 18 -adapter primers, and PCR was performed using two different sets of primers for both MeTHIC homologs. (C) Organization of the MeTHIC1 and MeTHIC2 3ʹ-UTR regions. CDS4 and CDS5 represent the last exons of the transcripts. An intron (intron 4) is located in front of the stop codon indicated by TGA and is spliced in transcripts Ib, II, and III. GU and AG identify the splice sites to obtain form III and the dashed lines indicate splicing events. The diamond indicates the transcript processing site. Primers used for PCR amplification of MeTHIC1 and MeTHIC2 3ʹ-UTR are indicated by arrows.
    Figure Legend Snippet: Organization of the cassava MeTHIC1 and MeTHIC2 3ʹ-UTR regions. PCR amplification of (A) MeTHIC1 and (B) MeTHIC2 3ʹ-UTR regions. Plants were grown in vitro in cassava basic medium without vitamin B 1 (indicated by 0) or in medium supplemented with 10 μM of vitamin B 1 (indicated by +B 1 ). cDNA was synthesized using oligo-(dT) 18 primers, random hexamer oligonucleotides, or oligo-(dT) 18 -adapter primers, and PCR was performed using two different sets of primers for both MeTHIC homologs. (C) Organization of the MeTHIC1 and MeTHIC2 3ʹ-UTR regions. CDS4 and CDS5 represent the last exons of the transcripts. An intron (intron 4) is located in front of the stop codon indicated by TGA and is spliced in transcripts Ib, II, and III. GU and AG identify the splice sites to obtain form III and the dashed lines indicate splicing events. The diamond indicates the transcript processing site. Primers used for PCR amplification of MeTHIC1 and MeTHIC2 3ʹ-UTR are indicated by arrows.

    Techniques Used: Polymerase Chain Reaction, Amplification, In Vitro, Synthesized, Random Hexamer Labeling

    22) Product Images from "3'-coterminal subgenomic RNAs and putative cis-acting elements of Grapevine leafroll-associated virus 3 reveals 'unique' features of gene expression strategy in the genus Ampelovirus"

    Article Title: 3'-coterminal subgenomic RNAs and putative cis-acting elements of Grapevine leafroll-associated virus 3 reveals 'unique' features of gene expression strategy in the genus Ampelovirus

    Journal: Virology Journal

    doi: 10.1186/1743-422X-7-180

    Northern blot analysis of total RNA extracted from grapevine (cv. Merlot) infected with GLRaV-3 . Northern blot hybridizations were carried out using a positive-stranded gene-specific riboprobes containing 3'terminus, p20A, p21, CPm, and CP sequences. Position of subgenomic (sg) RNAs is indicated by arrows on the left. Location of sgRNAs for CPm, p55 and HSP70h were tentative and indicated with an asterisk. The non-specific band present in all lanes is indicated by an arrow head.
    Figure Legend Snippet: Northern blot analysis of total RNA extracted from grapevine (cv. Merlot) infected with GLRaV-3 . Northern blot hybridizations were carried out using a positive-stranded gene-specific riboprobes containing 3'terminus, p20A, p21, CPm, and CP sequences. Position of subgenomic (sg) RNAs is indicated by arrows on the left. Location of sgRNAs for CPm, p55 and HSP70h were tentative and indicated with an asterisk. The non-specific band present in all lanes is indicated by an arrow head.

    Techniques Used: Northern Blot, Infection

    A schematic diagram of the GLRaV-3 genome . The ORFs, numbered as 1 to 12 above the diagram, are shown as boxes with associated protein designations. L-Pro, leader proteinase; AlkB, AlkB domain; MET, HEL, and POL, methyltransferase, RNA helicase, and RNA-dependent RNA polymerase domains of the replicase, respectively; p6, a 6-kDa protein; p5, a 5-kDa protein; HSP70 h, a HSP70-homologue; p55, a 55-kDa protein; CP, the major capsid protein; CPm, the minor capsid protein; and p21, p20A, p20B, p4 and p7 are the 21-, 19.6-, 19.7-, 4- and 7-kDa proteins, respectively. Below the genome map is a representation of (right) the 11 putative subgenomic messenger (m) RNAs for the 3' genes and (left) the polyproteins from ORFs 1a and 1b. The subgenomic mRNAs and their transcription start sites identified in this study are shown with an asterisk. Arrow head indicates site of +1 ribosomal frameshift.
    Figure Legend Snippet: A schematic diagram of the GLRaV-3 genome . The ORFs, numbered as 1 to 12 above the diagram, are shown as boxes with associated protein designations. L-Pro, leader proteinase; AlkB, AlkB domain; MET, HEL, and POL, methyltransferase, RNA helicase, and RNA-dependent RNA polymerase domains of the replicase, respectively; p6, a 6-kDa protein; p5, a 5-kDa protein; HSP70 h, a HSP70-homologue; p55, a 55-kDa protein; CP, the major capsid protein; CPm, the minor capsid protein; and p21, p20A, p20B, p4 and p7 are the 21-, 19.6-, 19.7-, 4- and 7-kDa proteins, respectively. Below the genome map is a representation of (right) the 11 putative subgenomic messenger (m) RNAs for the 3' genes and (left) the polyproteins from ORFs 1a and 1b. The subgenomic mRNAs and their transcription start sites identified in this study are shown with an asterisk. Arrow head indicates site of +1 ribosomal frameshift.

    Techniques Used:

    23) Product Images from "T-bet Deficiency Attenuates Renal Injury in Experimental Crescentic Glomerulonephritis"

    Article Title: T-bet Deficiency Attenuates Renal Injury in Experimental Crescentic Glomerulonephritis

    Journal:

    doi: 10.1681/ASN.2007030392

    Cytokine mRNA expression in spleens of mice 6 d after injection of sheep anti-mouse GBM globulin. T-bet −/− mice had a trend toward lower expression of IFN-γ (A; P = 0.06), no difference in IL-4 expression (B), and higher
    Figure Legend Snippet: Cytokine mRNA expression in spleens of mice 6 d after injection of sheep anti-mouse GBM globulin. T-bet −/− mice had a trend toward lower expression of IFN-γ (A; P = 0.06), no difference in IL-4 expression (B), and higher

    Techniques Used: Expressing, Mouse Assay, Injection

    Intrarenal mRNA expression of prototypical Th1 (A), Th2 (B), and Th17 (C) cytokines at day 21. Kidney mRNA expression of IFN-γ and IL-4 was unchanged in T-bet −/− mice, but expression of IL-17A was increased. * P
    Figure Legend Snippet: Intrarenal mRNA expression of prototypical Th1 (A), Th2 (B), and Th17 (C) cytokines at day 21. Kidney mRNA expression of IFN-γ and IL-4 was unchanged in T-bet −/− mice, but expression of IL-17A was increased. * P

    Techniques Used: Expressing, Mouse Assay

    24) Product Images from "Identification of Novel Inhibitors of the Type I Interferon Induction Pathway Using Cell-Based High-Throughput Screening"

    Article Title: Identification of Novel Inhibitors of the Type I Interferon Induction Pathway Using Cell-Based High-Throughput Screening

    Journal: Journal of Biomolecular Screening

    doi: 10.1177/1087057116656314

    Effect of StA-IFN-1 and StA-IFN-4 on interferon β (IFNβ) and MxA messenger RNA (mRNA) levels. Effect of StA-IFN-1, StA-IFN-4, and TPCA-1 on IFNβ mRNA levels in A549 cells infected with Sendai virus (SeV) ( A ) or StA-IFN-1, StA-IFN-4 and Ruxolitinib on MxA mRNA levels in A549 cells activated with purified interferon α (IFNα) ( B ). Cells were treated with compound 2 h prior to activation. Three hours post-SeV infection and 18 h post-IFNα treatment, total cellular RNA was extracted and reverse transcribed. The resultant complementary DNA (cDNA) was used to quantitative PCR amplify either IFNβ or MxA sequences using appropriate primers. C t values were subjected to absolute quantitation using a 6-point standard curve with DNA of known concentration and converted into % cDNA of controls. Data represent the mean of three independent experiments, each conducted in triplicate; error bars indicate SD. Statistical significance was assessed using the Student’s t test to compare compound treatment with the DMSO plus SeV or IFNα (* p
    Figure Legend Snippet: Effect of StA-IFN-1 and StA-IFN-4 on interferon β (IFNβ) and MxA messenger RNA (mRNA) levels. Effect of StA-IFN-1, StA-IFN-4, and TPCA-1 on IFNβ mRNA levels in A549 cells infected with Sendai virus (SeV) ( A ) or StA-IFN-1, StA-IFN-4 and Ruxolitinib on MxA mRNA levels in A549 cells activated with purified interferon α (IFNα) ( B ). Cells were treated with compound 2 h prior to activation. Three hours post-SeV infection and 18 h post-IFNα treatment, total cellular RNA was extracted and reverse transcribed. The resultant complementary DNA (cDNA) was used to quantitative PCR amplify either IFNβ or MxA sequences using appropriate primers. C t values were subjected to absolute quantitation using a 6-point standard curve with DNA of known concentration and converted into % cDNA of controls. Data represent the mean of three independent experiments, each conducted in triplicate; error bars indicate SD. Statistical significance was assessed using the Student’s t test to compare compound treatment with the DMSO plus SeV or IFNα (* p

    Techniques Used: Infection, Purification, Activation Assay, Real-time Polymerase Chain Reaction, Quantitation Assay, Concentration Assay

    25) Product Images from "The regulatory role of DR4 in a spontaneous diabetes DQ8 transgenic model"

    Article Title: The regulatory role of DR4 in a spontaneous diabetes DQ8 transgenic model

    Journal: Journal of Clinical Investigation

    doi:

    Expression of Ig isotypes in DQ8 + /mII – /RIP-B7, DR4 + /mII – /RIP-B7, and DQ8 + DR4 + /mII – /RIP-B7 mice. Serum levels of IgG1 and IgA were measured using ELISA (see Methods) in the three groups of mice as indicated. The number of mice in each group was 15, 20, and 22 for DQ8 + /mII – /RIP-B7, DR4 + /mII – /RIP-B7, and DQ8 + DR4 + /mII – /RIP-B7 mice, respectively. The results are presented as mean ± SD. Student’s t test was used for the statistical analysis.
    Figure Legend Snippet: Expression of Ig isotypes in DQ8 + /mII – /RIP-B7, DR4 + /mII – /RIP-B7, and DQ8 + DR4 + /mII – /RIP-B7 mice. Serum levels of IgG1 and IgA were measured using ELISA (see Methods) in the three groups of mice as indicated. The number of mice in each group was 15, 20, and 22 for DQ8 + /mII – /RIP-B7, DR4 + /mII – /RIP-B7, and DQ8 + DR4 + /mII – /RIP-B7 mice, respectively. The results are presented as mean ± SD. Student’s t test was used for the statistical analysis.

    Techniques Used: Expressing, Mouse Assay, Enzyme-linked Immunosorbent Assay

    Spontaneous diabetes development in HLA/RIP-B7 transgenic mice. Four groups of mice were used in the study as indicated. Number of mice per group was: n = 15 (7 females and 8 males) for DQ8 + /mII – /RIP-B7; n = 20 (11 females and 9 males) for DR4 + /mII – /RIP-B7; n = 22 (12 females and 10 males) for DQ8 + DR4 + /mII – /RIP-B7; n = 10 (4 females and 6 males) for mII – /RIP-B7 mice. Mice were housed in specific pathogen-free (SPF) conditions. Diabetes was determined by monitoring of urinary glucose and confirmed by blood glucose ( > 250 mg/dl).
    Figure Legend Snippet: Spontaneous diabetes development in HLA/RIP-B7 transgenic mice. Four groups of mice were used in the study as indicated. Number of mice per group was: n = 15 (7 females and 8 males) for DQ8 + /mII – /RIP-B7; n = 20 (11 females and 9 males) for DR4 + /mII – /RIP-B7; n = 22 (12 females and 10 males) for DQ8 + DR4 + /mII – /RIP-B7; n = 10 (4 females and 6 males) for mII – /RIP-B7 mice. Mice were housed in specific pathogen-free (SPF) conditions. Diabetes was determined by monitoring of urinary glucose and confirmed by blood glucose ( > 250 mg/dl).

    Techniques Used: Transgenic Assay, Mouse Assay

    Immunohistochemistry staining of pancreatic sections of diabetic mice, with the diabetic DQ8 + /mII – /RIP-B7 mouse in the upper panel, diabetic DR4 + /mII – /RIP-B7 mouse in the middle panel, and diabetic DQ8 + DR4 + /mII – /RIP-B7 mouse in the lower panel. Islet infiltrates were stained for CD4 + T cells (left), CD8 + T cells (middle), and B220 + B cells (right).
    Figure Legend Snippet: Immunohistochemistry staining of pancreatic sections of diabetic mice, with the diabetic DQ8 + /mII – /RIP-B7 mouse in the upper panel, diabetic DR4 + /mII – /RIP-B7 mouse in the middle panel, and diabetic DQ8 + DR4 + /mII – /RIP-B7 mouse in the lower panel. Islet infiltrates were stained for CD4 + T cells (left), CD8 + T cells (middle), and B220 + B cells (right).

    Techniques Used: Immunohistochemistry, Staining, Mouse Assay

    ( a ) Expression of HLA transgenes and the selection of CD4 + T cells in BM chimeras. In the top panel, lymphocytes isolated from the spleens of three types of chimeras (as indicated) were stained with the B-cell marker B220 (PE) in combination with either anti–HLA-DQ (FITC) or DR (FITC). In the lower panel, the same splenocytes were stained with anti-CD4 (PE) and anti-CD8 (FITC). ( b ) Immunohistochemistry staining of pancreatic sections of BM chimeras. In the upper panel are DQ8 + /mII – /RIP-B7 BM chimera. DR4 + /mII – /RIP-B7 BM chimera are in the middle panel. The lower panel shows DQ8 + DR4 + /mII – /RIP-B7 BM chimera. Islet infiltrates were stained for CD4 + T cells (left), CD8 + T cells (middle), and B220 + B cells (right).
    Figure Legend Snippet: ( a ) Expression of HLA transgenes and the selection of CD4 + T cells in BM chimeras. In the top panel, lymphocytes isolated from the spleens of three types of chimeras (as indicated) were stained with the B-cell marker B220 (PE) in combination with either anti–HLA-DQ (FITC) or DR (FITC). In the lower panel, the same splenocytes were stained with anti-CD4 (PE) and anti-CD8 (FITC). ( b ) Immunohistochemistry staining of pancreatic sections of BM chimeras. In the upper panel are DQ8 + /mII – /RIP-B7 BM chimera. DR4 + /mII – /RIP-B7 BM chimera are in the middle panel. The lower panel shows DQ8 + DR4 + /mII – /RIP-B7 BM chimera. Islet infiltrates were stained for CD4 + T cells (left), CD8 + T cells (middle), and B220 + B cells (right).

    Techniques Used: Expressing, Selection, Isolation, Staining, Marker, Immunohistochemistry

    Expression of HLA transgenes and the selection of CD4 + T cells. In the upper panel, lymphocytes isolated from peripheral blood in three types of mice (as indicated) were stained with the B-cell marker B220 (phycoerythrin; PE) in combination with either anti–HLA-DQ (FITC) or DR (FITC). In the middle panel, thymocytes were isolated from mice at 5–7 weeks of age and stained with anti-CD4 (PE; y axis) and anti-CD8 (FITC; x axis). Gated CD4 single-positive thymocytes were 5.2%, 5%, and 9.8% of total thymocytes analyzed in DQ8 + /mII – /RIP-B7, DR4 + /mII – /RIP-B7, and DQ8 + DR4 + /mII – /RIP-B7 mice, respectively. In the bottom panel, splenocytes (after removing erythrocytes) were stained with anti-CD4 (PE) and anti-CD8 (FITC). Gated CD4 single-positive splenocytes were 11.4%, 11.8%, and 19.5% of total splenocytes analyzed in DQ8 + /mII – /RIP-B7, DR4 + /mII – /RIP-B7, and DQ8 + DR4 + /mII – /RIP-B7 mice, respectively.
    Figure Legend Snippet: Expression of HLA transgenes and the selection of CD4 + T cells. In the upper panel, lymphocytes isolated from peripheral blood in three types of mice (as indicated) were stained with the B-cell marker B220 (phycoerythrin; PE) in combination with either anti–HLA-DQ (FITC) or DR (FITC). In the middle panel, thymocytes were isolated from mice at 5–7 weeks of age and stained with anti-CD4 (PE; y axis) and anti-CD8 (FITC; x axis). Gated CD4 single-positive thymocytes were 5.2%, 5%, and 9.8% of total thymocytes analyzed in DQ8 + /mII – /RIP-B7, DR4 + /mII – /RIP-B7, and DQ8 + DR4 + /mII – /RIP-B7 mice, respectively. In the bottom panel, splenocytes (after removing erythrocytes) were stained with anti-CD4 (PE) and anti-CD8 (FITC). Gated CD4 single-positive splenocytes were 11.4%, 11.8%, and 19.5% of total splenocytes analyzed in DQ8 + /mII – /RIP-B7, DR4 + /mII – /RIP-B7, and DQ8 + DR4 + /mII – /RIP-B7 mice, respectively.

    Techniques Used: Expressing, Selection, Isolation, Mouse Assay, Staining, Marker

    26) Product Images from "MiR-142-3p functions as a potential tumor suppressor directly targeting HMGB1 in non-small-cell lung carcinoma"

    Article Title: MiR-142-3p functions as a potential tumor suppressor directly targeting HMGB1 in non-small-cell lung carcinoma

    Journal: International Journal of Clinical and Experimental Pathology

    doi:

    MiR-142-3p directly targets HMGB1 mRNA and inhibits its expression. A. The putative miR-142-3p-binding sites in the 3’-UTR of HMGB1 mRNA was shown. Mutation was generated on the HMGB1 3’-UTR sequence in the complementary site for the seed
    Figure Legend Snippet: MiR-142-3p directly targets HMGB1 mRNA and inhibits its expression. A. The putative miR-142-3p-binding sites in the 3’-UTR of HMGB1 mRNA was shown. Mutation was generated on the HMGB1 3’-UTR sequence in the complementary site for the seed

    Techniques Used: Expressing, Binding Assay, Mutagenesis, Generated, Sequencing

    27) Product Images from "Translational Control of Mitochondrial Energy Production Mediates Neuron Morphogenesis"

    Article Title: Translational Control of Mitochondrial Energy Production Mediates Neuron Morphogenesis

    Journal: Cell metabolism

    doi: 10.1016/j.cmet.2012.11.002

    CPEB1 Regulates NDUFV2 mRNA Translation (A) Schematic of NDUFV2 mRNA showing the mitochondrial targeting signal, polyadenylation hexanucleotide AAUAAA, and CPEs. (B) ATP was measured in WT and CPEB1 KO hippocampal neurons infected with lentiviruses expressing NDUFV2, or a mutant NDUFV2 lacking the MTS or NDUFS4. (C) Complex I activity was measured in WT and CPEB1 KO hippocampal neurons infected with lentivirus expressing NDUFV2. (D) WT hippocampal neurons were infected with lentivirus expressing HA-CPEB1. HA antibody was used to coimmunoprecipitate CPEB1 followed by RT-PCR for NDUFV2, NDUFS4, and GAPDH mRNAs. (E) WT and CPEB1 KO hippocampal neurons were incubated with 35 S-methionine followed by immunoprecipitation of NDUFV2 and analysis by SDS-PAGE. WCE refers to whole cell extract. The histogram shows the quantification of immunoprecipitation. (F) The coding region of Renilla luciferase was appended with the WT NDUFV2 3′UTR or one that lacked the CPEs (3′UTR mut) (see A). Plasmids encoding these constructs as well as firefly luciferase to serve as a control were transfected into neurons and analyzed for luciferase activity 3 days later. The data are plotted as the ratio of Renilla luciferase activity to firefly luciferase activity. (G) A PCR-based polyadenylation assay was used to determine the poly(A) tails of NDUFV2 and tubulin mRNAs in WT and CPEB1 KO brain (primers a/dT anchor). The internal primers (a/b) indicate the relative amount of RNA in each sample. *p
    Figure Legend Snippet: CPEB1 Regulates NDUFV2 mRNA Translation (A) Schematic of NDUFV2 mRNA showing the mitochondrial targeting signal, polyadenylation hexanucleotide AAUAAA, and CPEs. (B) ATP was measured in WT and CPEB1 KO hippocampal neurons infected with lentiviruses expressing NDUFV2, or a mutant NDUFV2 lacking the MTS or NDUFS4. (C) Complex I activity was measured in WT and CPEB1 KO hippocampal neurons infected with lentivirus expressing NDUFV2. (D) WT hippocampal neurons were infected with lentivirus expressing HA-CPEB1. HA antibody was used to coimmunoprecipitate CPEB1 followed by RT-PCR for NDUFV2, NDUFS4, and GAPDH mRNAs. (E) WT and CPEB1 KO hippocampal neurons were incubated with 35 S-methionine followed by immunoprecipitation of NDUFV2 and analysis by SDS-PAGE. WCE refers to whole cell extract. The histogram shows the quantification of immunoprecipitation. (F) The coding region of Renilla luciferase was appended with the WT NDUFV2 3′UTR or one that lacked the CPEs (3′UTR mut) (see A). Plasmids encoding these constructs as well as firefly luciferase to serve as a control were transfected into neurons and analyzed for luciferase activity 3 days later. The data are plotted as the ratio of Renilla luciferase activity to firefly luciferase activity. (G) A PCR-based polyadenylation assay was used to determine the poly(A) tails of NDUFV2 and tubulin mRNAs in WT and CPEB1 KO brain (primers a/dT anchor). The internal primers (a/b) indicate the relative amount of RNA in each sample. *p

    Techniques Used: Infection, Expressing, Mutagenesis, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Incubation, Immunoprecipitation, SDS Page, Luciferase, Construct, Transfection, Polymerase Chain Reaction

    28) Product Images from "Identification, Purification, and Molecular Cloning of a Putative Plastidic Glucose Translocator"

    Article Title: Identification, Purification, and Molecular Cloning of a Putative Plastidic Glucose Translocator

    Journal: The Plant Cell

    doi:

    Expression Analysis of the Gene Encoding pGlcT. (A) RNA gel blot analysis of pGlcT mRNA in different tissues of tobacco. Thirty micrograms of total RNA isolated from source leaves, stems, sink leaves, roots, flower buds, and petioles was hybridized with the pGlcT cDNA probe from spinach after gel electrophoresis and subsequent transfer of the RNA to a nylon membrane. (B) Diurnal variation of the steady state concentration of pGlcT mRNA in source leaves of tobacco. Total RNA was isolated from source leaves of tobacco plants after 10 hr of light (lane 1), 1 hr of dark (lane 2), 4 hr of dark (lane 3), 12 hr of dark (lane 4), 1 hr of light (lane 5), and 3 hr of light (lane 6) during a 12-hr-light/12-hr-dark cycle. Analysis (30 μg per lane) was performed as indicated in (A) . (C) ). Analysis (50 μg per lane) was performed as in (A) .
    Figure Legend Snippet: Expression Analysis of the Gene Encoding pGlcT. (A) RNA gel blot analysis of pGlcT mRNA in different tissues of tobacco. Thirty micrograms of total RNA isolated from source leaves, stems, sink leaves, roots, flower buds, and petioles was hybridized with the pGlcT cDNA probe from spinach after gel electrophoresis and subsequent transfer of the RNA to a nylon membrane. (B) Diurnal variation of the steady state concentration of pGlcT mRNA in source leaves of tobacco. Total RNA was isolated from source leaves of tobacco plants after 10 hr of light (lane 1), 1 hr of dark (lane 2), 4 hr of dark (lane 3), 12 hr of dark (lane 4), 1 hr of light (lane 5), and 3 hr of light (lane 6) during a 12-hr-light/12-hr-dark cycle. Analysis (30 μg per lane) was performed as indicated in (A) . (C) ). Analysis (50 μg per lane) was performed as in (A) .

    Techniques Used: Expressing, Western Blot, Isolation, Nucleic Acid Electrophoresis, Concentration Assay

    29) Product Images from "Full-Length Enriched cDNA Libraries and ORFeome Analysis of Sugarcane Hybrid and Ancestor Genotypes"

    Article Title: Full-Length Enriched cDNA Libraries and ORFeome Analysis of Sugarcane Hybrid and Ancestor Genotypes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0107351

    Full-length enrichment for library cloning and next generation sequencing (NGS). Full-length (blue line with 5′ cap) or truncated (short blue line without 5′ cap) mRNAs were reverse transcribed into first-strand cDNA using oligo-dT primers (red arrow). The mRNA:cDNA hybrid was treated with RNase I (scissor) to remove the single-stranded RNA that was not fully extended by the first-strand cDNA, followed by selection for full-length transcripts using Cap-antibody magnetic beads to enrich the full-length mRNA:cDNA. The full-length single-stranded DNA (FLssDNA) was eluted from beads and used for both cDNA library cloning (lower left) and NGS (lower right). For full-length library cloning, a double-stranded adaptor (green) was linked to the 5′ end of ssDNA. Second-strand cDNA synthesis was then carried out, followed by cloning into a vector. For NGS, the full-length enriched ssDNA was fragmented by sonication to target fragments in the range of 200–400 bp, followed by ligation of the double-stranded DNA sequencing adaptor mixture (purple) to 3′ and 5′ ends of ssDNA. To maintain the complexity of the library while enriching the full-length cDNA for NGS, the original polyA mRNA was also fragmented using RNAse III, followed by ligation of the double-stranded RNA sequencing adaptor mixture (brown) to 3′ and 5′ ends of mRNA. After first- and second-strand synthesis, the polyA and capped mRNA and polyA and non-capped mRNA samples were mixed in a 3∶1 ratio and applied to the downstream NGS procedure.
    Figure Legend Snippet: Full-length enrichment for library cloning and next generation sequencing (NGS). Full-length (blue line with 5′ cap) or truncated (short blue line without 5′ cap) mRNAs were reverse transcribed into first-strand cDNA using oligo-dT primers (red arrow). The mRNA:cDNA hybrid was treated with RNase I (scissor) to remove the single-stranded RNA that was not fully extended by the first-strand cDNA, followed by selection for full-length transcripts using Cap-antibody magnetic beads to enrich the full-length mRNA:cDNA. The full-length single-stranded DNA (FLssDNA) was eluted from beads and used for both cDNA library cloning (lower left) and NGS (lower right). For full-length library cloning, a double-stranded adaptor (green) was linked to the 5′ end of ssDNA. Second-strand cDNA synthesis was then carried out, followed by cloning into a vector. For NGS, the full-length enriched ssDNA was fragmented by sonication to target fragments in the range of 200–400 bp, followed by ligation of the double-stranded DNA sequencing adaptor mixture (purple) to 3′ and 5′ ends of ssDNA. To maintain the complexity of the library while enriching the full-length cDNA for NGS, the original polyA mRNA was also fragmented using RNAse III, followed by ligation of the double-stranded RNA sequencing adaptor mixture (brown) to 3′ and 5′ ends of mRNA. After first- and second-strand synthesis, the polyA and capped mRNA and polyA and non-capped mRNA samples were mixed in a 3∶1 ratio and applied to the downstream NGS procedure.

    Techniques Used: Clone Assay, Next-Generation Sequencing, Selection, Magnetic Beads, cDNA Library Assay, Plasmid Preparation, Sonication, Ligation, DNA Sequencing, RNA Sequencing Assay

    30) Product Images from "Differential Expression Analysis of Olfactory Genes Based on a Combination of Sequencing Platforms and Behavioral Investigations in Aphidius gifuensis"

    Article Title: Differential Expression Analysis of Olfactory Genes Based on a Combination of Sequencing Platforms and Behavioral Investigations in Aphidius gifuensis

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2018.01679

    Venn diagram based on a combined analysis of differential expression of transcripts and behavioral investigations. (A,B) According to the behavioral test results for the response to wasps of the opposite sex, the intersection of differentially expressed olfactory genes in VM/VF and comparable olfactory genes in MM/VF contains five genes (upregulated) and one gene (downregulated), which represent six candidate genes that may be involved in the recognition of the opposite sex by VMs. The intersection of differentially expressed olfactory genes in MF/VF and comparable olfactory genes in MM/VF contains three genes (upregulated) and four genes (downregulated), respectively, which represents seven candidate genes that could be involved in the recognition of the opposite sex by MFs. (C,D) According to the results of the behavioral test for the response to EBF, the number of common up- and downregulated olfactory genes was 1 and 3, respectively, which represent four candidate genes that may be involved in EBF perception.
    Figure Legend Snippet: Venn diagram based on a combined analysis of differential expression of transcripts and behavioral investigations. (A,B) According to the behavioral test results for the response to wasps of the opposite sex, the intersection of differentially expressed olfactory genes in VM/VF and comparable olfactory genes in MM/VF contains five genes (upregulated) and one gene (downregulated), which represent six candidate genes that may be involved in the recognition of the opposite sex by VMs. The intersection of differentially expressed olfactory genes in MF/VF and comparable olfactory genes in MM/VF contains three genes (upregulated) and four genes (downregulated), respectively, which represents seven candidate genes that could be involved in the recognition of the opposite sex by MFs. (C,D) According to the results of the behavioral test for the response to EBF, the number of common up- and downregulated olfactory genes was 1 and 3, respectively, which represent four candidate genes that may be involved in EBF perception.

    Techniques Used: Expressing

    Heatmap of all the annotated olfactory genes. Genes marked in green, three of six candidate genes involved in recognition of the opposite sex by VMs; blue, six candidate genes involved in recognition of the opposite sex by MFs; red, the remaining four of seven candidate genes involved in recognition of the opposite sex by VMs, which are also involved in EBF perception.
    Figure Legend Snippet: Heatmap of all the annotated olfactory genes. Genes marked in green, three of six candidate genes involved in recognition of the opposite sex by VMs; blue, six candidate genes involved in recognition of the opposite sex by MFs; red, the remaining four of seven candidate genes involved in recognition of the opposite sex by VMs, which are also involved in EBF perception.

    Techniques Used:

    31) Product Images from "Isolation of an FMRP-Associated Messenger Ribonucleoprotein Particle and Identification of Nucleolin and the Fragile X-Related Proteins as Components of the Complex"

    Article Title: Isolation of an FMRP-Associated Messenger Ribonucleoprotein Particle and Identification of Nucleolin and the Fragile X-Related Proteins as Components of the Complex

    Journal: Molecular and Cellular Biology

    doi:

    FXR1P and FXR2P assemble with Flag-FMRP in transfected L-M(TK−) cells to form an mRNP particle that binds mRNA. (A) Cytoplasmic lysates from approximately 5 × 10 5 L-M(TK−) cells expressing vector alone and expressing Flag-FMRP were loaded into lanes 1 and 2, respectively; lanes 3 to 5 contain Flag peptide elutions from the anti-Flag antibody M2 alone (lane 3) or from immunoprecipitations of 10 7 cells expressing the vector -only (lane 4) or Flag-FMRP (lane 5). The immunoprecipitated FMRP in lane 5 appears to run slower than the FMRP detected in the cytoplasmic lysates, probably because there is much less protein in the lanes containing the peptide elutions than in the lanes containing cytoplasmic lysates. The gel was blotted and sequentially probed with a monoclonal antibody recognizing FMRP (A), then with both anti-FMRP and anti-FXR2P antibodies (B), and finally with anti-FMRP, anti-FXR2P and anti-FXR1P antibodies (C). Lanes 1 and 2 in panel C are shown as separate because they are a lighter exposure of the same blot. Positions of the molecular weight standards are shown on the left, and positions of the proteins are shown on the right. The long and short isoforms of FXR1P are indicated by lines. (D) mRNA was purified from L-M(TK−) cells expressing either the vector only (lane 1) or Flag-FMRP (lane 2) as described in Materials and Methods. The mRNA was recovered, and the polyadenylated species were labeled by priming with oligo(dT) and synthesizing first-strand cDNA with reverse transcriptase. (E) MRNA obtained from immunoprecipitations of L-M(TK−) cells expressing either Flag-FMRP (lanes 1 and 2) or vector alone (lanes 3 and 4) or from mouse brain (lanes 5 and 6) was reverse transcribed with an oligo(dT) primer in either the presence (lanes 2, 4, and 6) or the absence (lanes 1, 3, and 5) of reverse transcriptase. A fraction of each reaction mixture was then added to a PCR mixture with mouse FMR1 primers. The PCR products were resolved on an agarose gel and stained with ethidium bromide.
    Figure Legend Snippet: FXR1P and FXR2P assemble with Flag-FMRP in transfected L-M(TK−) cells to form an mRNP particle that binds mRNA. (A) Cytoplasmic lysates from approximately 5 × 10 5 L-M(TK−) cells expressing vector alone and expressing Flag-FMRP were loaded into lanes 1 and 2, respectively; lanes 3 to 5 contain Flag peptide elutions from the anti-Flag antibody M2 alone (lane 3) or from immunoprecipitations of 10 7 cells expressing the vector -only (lane 4) or Flag-FMRP (lane 5). The immunoprecipitated FMRP in lane 5 appears to run slower than the FMRP detected in the cytoplasmic lysates, probably because there is much less protein in the lanes containing the peptide elutions than in the lanes containing cytoplasmic lysates. The gel was blotted and sequentially probed with a monoclonal antibody recognizing FMRP (A), then with both anti-FMRP and anti-FXR2P antibodies (B), and finally with anti-FMRP, anti-FXR2P and anti-FXR1P antibodies (C). Lanes 1 and 2 in panel C are shown as separate because they are a lighter exposure of the same blot. Positions of the molecular weight standards are shown on the left, and positions of the proteins are shown on the right. The long and short isoforms of FXR1P are indicated by lines. (D) mRNA was purified from L-M(TK−) cells expressing either the vector only (lane 1) or Flag-FMRP (lane 2) as described in Materials and Methods. The mRNA was recovered, and the polyadenylated species were labeled by priming with oligo(dT) and synthesizing first-strand cDNA with reverse transcriptase. (E) MRNA obtained from immunoprecipitations of L-M(TK−) cells expressing either Flag-FMRP (lanes 1 and 2) or vector alone (lanes 3 and 4) or from mouse brain (lanes 5 and 6) was reverse transcribed with an oligo(dT) primer in either the presence (lanes 2, 4, and 6) or the absence (lanes 1, 3, and 5) of reverse transcriptase. A fraction of each reaction mixture was then added to a PCR mixture with mouse FMR1 primers. The PCR products were resolved on an agarose gel and stained with ethidium bromide.

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Molecular Weight, Purification, Labeling, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining

    32) Product Images from "Characterization of the Transcription Profile of Adeno-Associated Virus Type 5 Reveals a Number of Unique Features Compared to Previously Characterized Adeno-Associated Viruses"

    Article Title: Characterization of the Transcription Profile of Adeno-Associated Virus Type 5 Reveals a Number of Unique Features Compared to Previously Characterized Adeno-Associated Viruses

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.24.12435-12447.2002

    (A) Splicing of AAV5 RNA is independent of Ad5 and Rep. 293 cells were either infected with AAV5 virus (15 IU/cell) (lanes 1 and 2) or transfected, in the presence (+) or absence (−) of Ad5 (5 PFU/cell), with the following plasmids, which are diagramed on the left: a replicating full-length AAV5 genomic plasmid (pAV5) (lanes 3 and 4); an AAV5 plasmid which contains the AAV5 rep and cap genes and lacks the ITRs (pAV5RepCap) (lanes 5 and 6); a full-length plasmid, pAV5Δ752C, that produces a mutant Rep protein (lanes 7 and 8); pAV5Δ752C cotransfected with a Rep-supplementing plasmid pHIVAV5RepSM (lanes 9 and 10); or an AAV2 rep and cap gene-containing plasmid (pAV2RepCap) (lanes 11 and 12). Total RNA (10 μg), taken 36 to 40 h after transfection or infection, was protected by either the AAV5 RP or AAV2 RP probe, and the gel from a representative experiment is shown. The identities of the bands are shown on the right. The ratio of spliced P41 (or P40) transcripts to transcripts that are either unspliced or polyadenylated at the proximal site [(pA)p] are shown and are averages of theresults of at least three separate experiments with standard deviations. The arrow and arrowhead show unspliced RNA and RNA utilizing (pA)d, or spliced RNA, respectively, generated from promoters upstream of P41. (B) 293 cells were transfected with the AAV5 plasmids diagramed on the left, either with (Rep+) or without (Rep−) cotransfection of the AAV5 Rep-supplementing plasmid (pHIVAV5RepSM) in the presence of Ad5 coinfection (5 PFU/cell). Total RNA (10 μg), taken 36 to 40 h posttransfection, was protected by the RP probe, and the image shown is from a representative experiment. The ratio of spliced P41 transcripts to transcripts that are either unspliced or polyadenylated at the proximal site [(pA)p] are shown and are averages of the results of at least three separate experiments with standard deviations. The arrow and arrowhead show unspliced RNA and RNA utilizing the proximal polyadenylation site [(pA)p], or spliced RNA, respectively, generated from promoters upstream of P41.
    Figure Legend Snippet: (A) Splicing of AAV5 RNA is independent of Ad5 and Rep. 293 cells were either infected with AAV5 virus (15 IU/cell) (lanes 1 and 2) or transfected, in the presence (+) or absence (−) of Ad5 (5 PFU/cell), with the following plasmids, which are diagramed on the left: a replicating full-length AAV5 genomic plasmid (pAV5) (lanes 3 and 4); an AAV5 plasmid which contains the AAV5 rep and cap genes and lacks the ITRs (pAV5RepCap) (lanes 5 and 6); a full-length plasmid, pAV5Δ752C, that produces a mutant Rep protein (lanes 7 and 8); pAV5Δ752C cotransfected with a Rep-supplementing plasmid pHIVAV5RepSM (lanes 9 and 10); or an AAV2 rep and cap gene-containing plasmid (pAV2RepCap) (lanes 11 and 12). Total RNA (10 μg), taken 36 to 40 h after transfection or infection, was protected by either the AAV5 RP or AAV2 RP probe, and the gel from a representative experiment is shown. The identities of the bands are shown on the right. The ratio of spliced P41 (or P40) transcripts to transcripts that are either unspliced or polyadenylated at the proximal site [(pA)p] are shown and are averages of theresults of at least three separate experiments with standard deviations. The arrow and arrowhead show unspliced RNA and RNA utilizing (pA)d, or spliced RNA, respectively, generated from promoters upstream of P41. (B) 293 cells were transfected with the AAV5 plasmids diagramed on the left, either with (Rep+) or without (Rep−) cotransfection of the AAV5 Rep-supplementing plasmid (pHIVAV5RepSM) in the presence of Ad5 coinfection (5 PFU/cell). Total RNA (10 μg), taken 36 to 40 h posttransfection, was protected by the RP probe, and the image shown is from a representative experiment. The ratio of spliced P41 transcripts to transcripts that are either unspliced or polyadenylated at the proximal site [(pA)p] are shown and are averages of the results of at least three separate experiments with standard deviations. The arrow and arrowhead show unspliced RNA and RNA utilizing the proximal polyadenylation site [(pA)p], or spliced RNA, respectively, generated from promoters upstream of P41.

    Techniques Used: Infection, Transfection, Plasmid Preparation, Mutagenesis, Generated, Cotransfection

    (A) Schematic diagram of the AAV genome and the probes used in this study. The ITRs, the promoters (P7, P19, and P41), the intron donor (D) and acceptors (A1 and A2), and the proximal [(pA)p] and distal [(pA)d] polyadenylation sites are shown. The locations of the IP (nt 269 to 480), SB (nt 801 to 1023), RP (nt 1843 to 2034), DH (nt 1994 to 2341), and PA (nt 4294 to 4498) probes used in this study are shown. Below each probe is a depiction of the bands expected following protection by each of the designated RNA species. (B) Mapping of the AAV5 transcription units by RPA. 293 cells were infected with AAV5 (15 IU/cell) and Ad5 (5 PFU/cell). Ten micrograms of total RNA was isolated 36 to 40 h after coinfection and protected by the IP, SB, RP, DH, and PA probes as indicated. Lane 1, a 32 P-labeled RNA ladder with the respective sizes indicated to the left. Lane 2, protection of total RNA generated following AAV2 infection of 293 cells by a homologous RP probe which serves as an additional marker. The sizes of these bands are 192, 139, 105, and 53 nt, respectively. The origins of the protected bands in lanes 3, 4, 6, and 7 are indicated. For lane 5, the top arrow designates unspliced and proximally polyadenylated [(pA)p] RNAs from the upstream promoter within the ITR (Inr), P7, and P19 (top), and the lower arrow indicates unspliced RNA and RNAs utilizing (pA)p generated from the P41 promoter. The top arrowhead shows spliced RNA from P7 plus P19, and perhaps from the ITR Inr, and the lower arrowhead identifies bands protected by spliced P41 RNA. The band designated by a star in lane 3 is undigested probe. The band designated by a diamond in lane 7 is likely transcription through the distal polyadenylation site (pA)d.
    Figure Legend Snippet: (A) Schematic diagram of the AAV genome and the probes used in this study. The ITRs, the promoters (P7, P19, and P41), the intron donor (D) and acceptors (A1 and A2), and the proximal [(pA)p] and distal [(pA)d] polyadenylation sites are shown. The locations of the IP (nt 269 to 480), SB (nt 801 to 1023), RP (nt 1843 to 2034), DH (nt 1994 to 2341), and PA (nt 4294 to 4498) probes used in this study are shown. Below each probe is a depiction of the bands expected following protection by each of the designated RNA species. (B) Mapping of the AAV5 transcription units by RPA. 293 cells were infected with AAV5 (15 IU/cell) and Ad5 (5 PFU/cell). Ten micrograms of total RNA was isolated 36 to 40 h after coinfection and protected by the IP, SB, RP, DH, and PA probes as indicated. Lane 1, a 32 P-labeled RNA ladder with the respective sizes indicated to the left. Lane 2, protection of total RNA generated following AAV2 infection of 293 cells by a homologous RP probe which serves as an additional marker. The sizes of these bands are 192, 139, 105, and 53 nt, respectively. The origins of the protected bands in lanes 3, 4, 6, and 7 are indicated. For lane 5, the top arrow designates unspliced and proximally polyadenylated [(pA)p] RNAs from the upstream promoter within the ITR (Inr), P7, and P19 (top), and the lower arrow indicates unspliced RNA and RNAs utilizing (pA)p generated from the P41 promoter. The top arrowhead shows spliced RNA from P7 plus P19, and perhaps from the ITR Inr, and the lower arrowhead identifies bands protected by spliced P41 RNA. The band designated by a star in lane 3 is undigested probe. The band designated by a diamond in lane 7 is likely transcription through the distal polyadenylation site (pA)d.

    Techniques Used: Recombinase Polymerase Amplification, Infection, Isolation, Labeling, Generated, Marker

    (A) AAV5 utilizes a polyadenylation site within the intron. Left panel, RT-PCR of polyadenylated RNA isolated from either uninfected 293 cells (lanes 1, 3, 5, and 7) or 293 cells infected with both AAV5 (15 IU/cell) and Ad5 (5 PFU/cell) (lanes 2, 4, 6, and 8), using an oligo(dT) 15 primer (dT15) and one of four forward primers: F1843 (lanes 1 and 2), F1912 (lanes 3 and 4), F1998 (lanes 5 and 6), and F4250 (lanes 7 and 8). The positions of the primers are diagrammed above the gel. The sizes of DNA fragments in the 100-bp ladder (Promega) (lane 9) are 1,500, 1,000, 900, 800, 700, 600, 500, 400, 300, 200, and 100 bp. The sizes of the DNA fragments in the 1-kb ladder (Promega) (lane 10) are 10, 8, 6, 5, 4, 3, 2.5, 2, 1.5, 1, 0.75, 0.5, and 0.25 kb. Expected sizes of the PCR products for transcripts using the proximal poly(A) site (pA)p, including the poly(A) tail, are shown above the gel. The PCR conditions used did not efficiently amplify the longer RNA products using the distal polyadenylation site. Right panel, Total RNA (1 μg) from AAV5-infected 293 cells was subjected to anchored RT-PCR using oligo(dT)19V (V = A, G, or C) (dT19V), both for the first-strand cDNA synthesis and with a forward primer for subsequent PCR. Two different forward primers, F1843, which starts at nt 1843, and F1882, which starts at nt 1882 (diagrammed above the gel), were used and resulted in bands of different mobilities (compare lane 11 with lane 12). Sequencing of the PCR products from lanes 11 and 12 yielded an identical sequence prior to the U at nt 2293. However, because there are three A residues after the U at nt 2193, the cleavage site may range from nt 2193 to 2196 (diagramed at the bottom). (B) Left panel, a PCR product containing only AAV5 DNA nt 185 to 2320 was transfected into 293 cells in the presence of Ad5 (5 PFU/cell), and total RNA (10 μg) from transfected cells was protected by the RP (lane 1) and DH (lane 2) probes. The designations of the protected bands are shown. Right panel, immunoblot analysis of AAV5 rep gene products in cell lysates of 293 cells infected with AAV5 (15 IU/cell) plus Ad5 (5 PFU/cell) (lane 1) or transfected with an AAV5 PCR product comprising nt 185 to 2320 (5 μg/60-mm-diameter dish) with Ad5 coinfection (lane 3), infected with AAV2 (10 IU/cell) plus Ad5 (lane 5), or infected with Ad5 alone as a negative control (lanes 2 and 4). A monoclonal antibody raised to NH 2 -terminally truncated AAV2 Rep proteins (monoclonal antibody 303.9; American Research Products) was used to identify AAV2 and AAV5 Rep proteins. Binding of 303.9 to the blot was detected by using a horseradish peroxidase-conjugated goat anti-mouse antibody. The migration of the AAV5 Rep protein products was compared to that of a series of prestained proteins (Invitrogen) The positions and apparent molecular masses of the markers are shown on the right of the immunoblot. Lane 5 shows the AAV2 Rep proteins, Rep78, -68, -52, and -40. The arrow and arrowhead identify AAV5 Rep78 and -52, respectively. The band designated by the star is most likely a degradation product, as its abundance increases upon sample storage; its derivation is not known.
    Figure Legend Snippet: (A) AAV5 utilizes a polyadenylation site within the intron. Left panel, RT-PCR of polyadenylated RNA isolated from either uninfected 293 cells (lanes 1, 3, 5, and 7) or 293 cells infected with both AAV5 (15 IU/cell) and Ad5 (5 PFU/cell) (lanes 2, 4, 6, and 8), using an oligo(dT) 15 primer (dT15) and one of four forward primers: F1843 (lanes 1 and 2), F1912 (lanes 3 and 4), F1998 (lanes 5 and 6), and F4250 (lanes 7 and 8). The positions of the primers are diagrammed above the gel. The sizes of DNA fragments in the 100-bp ladder (Promega) (lane 9) are 1,500, 1,000, 900, 800, 700, 600, 500, 400, 300, 200, and 100 bp. The sizes of the DNA fragments in the 1-kb ladder (Promega) (lane 10) are 10, 8, 6, 5, 4, 3, 2.5, 2, 1.5, 1, 0.75, 0.5, and 0.25 kb. Expected sizes of the PCR products for transcripts using the proximal poly(A) site (pA)p, including the poly(A) tail, are shown above the gel. The PCR conditions used did not efficiently amplify the longer RNA products using the distal polyadenylation site. Right panel, Total RNA (1 μg) from AAV5-infected 293 cells was subjected to anchored RT-PCR using oligo(dT)19V (V = A, G, or C) (dT19V), both for the first-strand cDNA synthesis and with a forward primer for subsequent PCR. Two different forward primers, F1843, which starts at nt 1843, and F1882, which starts at nt 1882 (diagrammed above the gel), were used and resulted in bands of different mobilities (compare lane 11 with lane 12). Sequencing of the PCR products from lanes 11 and 12 yielded an identical sequence prior to the U at nt 2293. However, because there are three A residues after the U at nt 2193, the cleavage site may range from nt 2193 to 2196 (diagramed at the bottom). (B) Left panel, a PCR product containing only AAV5 DNA nt 185 to 2320 was transfected into 293 cells in the presence of Ad5 (5 PFU/cell), and total RNA (10 μg) from transfected cells was protected by the RP (lane 1) and DH (lane 2) probes. The designations of the protected bands are shown. Right panel, immunoblot analysis of AAV5 rep gene products in cell lysates of 293 cells infected with AAV5 (15 IU/cell) plus Ad5 (5 PFU/cell) (lane 1) or transfected with an AAV5 PCR product comprising nt 185 to 2320 (5 μg/60-mm-diameter dish) with Ad5 coinfection (lane 3), infected with AAV2 (10 IU/cell) plus Ad5 (lane 5), or infected with Ad5 alone as a negative control (lanes 2 and 4). A monoclonal antibody raised to NH 2 -terminally truncated AAV2 Rep proteins (monoclonal antibody 303.9; American Research Products) was used to identify AAV2 and AAV5 Rep proteins. Binding of 303.9 to the blot was detected by using a horseradish peroxidase-conjugated goat anti-mouse antibody. The migration of the AAV5 Rep protein products was compared to that of a series of prestained proteins (Invitrogen) The positions and apparent molecular masses of the markers are shown on the right of the immunoblot. Lane 5 shows the AAV2 Rep proteins, Rep78, -68, -52, and -40. The arrow and arrowhead identify AAV5 Rep78 and -52, respectively. The band designated by the star is most likely a degradation product, as its abundance increases upon sample storage; its derivation is not known.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Isolation, Infection, Polymerase Chain Reaction, Sequencing, Transfection, Negative Control, Binding Assay, Migration

    33) Product Images from "HspB1 silences translation of PDZ-RhoGEF by enhancing miR-20a and miR-128 expression to promote neurite extension"

    Article Title: HspB1 silences translation of PDZ-RhoGEF by enhancing miR-20a and miR-128 expression to promote neurite extension

    Journal: Molecular and cellular neurosciences

    doi: 10.1016/j.mcn.2013.10.006

    HspB1 inhibits RhoA activity and reduces PDZ-RhoGEF protein levels
    Figure Legend Snippet: HspB1 inhibits RhoA activity and reduces PDZ-RhoGEF protein levels

    Techniques Used: Activity Assay

    Reduction in PDZ-RhoGEF by HspB1 is independent of protein degradation or transcriptional regulation
    Figure Legend Snippet: Reduction in PDZ-RhoGEF by HspB1 is independent of protein degradation or transcriptional regulation

    Techniques Used:

    HspB1 decreases PDZ-RhoGEF through regulation of miR-20a and miR-128
    Figure Legend Snippet: HspB1 decreases PDZ-RhoGEF through regulation of miR-20a and miR-128

    Techniques Used:

    34) Product Images from "Hormonal regulation of mannan-binding lectin synthesis in hepatocytes"

    Article Title: Hormonal regulation of mannan-binding lectin synthesis in hepatocytes

    Journal:

    doi: 10.1111/j.1365-2249.2006.03101.x

    The association between mannan-binding lectin (MBL) levels and total thyroid hormone T4 (TT4) in 17 healthy individuals with identical MBL2 genotype. TT4 levels are given on the abscissa and the corresponding MBL levels on the ordinate. The line drawn
    Figure Legend Snippet: The association between mannan-binding lectin (MBL) levels and total thyroid hormone T4 (TT4) in 17 healthy individuals with identical MBL2 genotype. TT4 levels are given on the abscissa and the corresponding MBL levels on the ordinate. The line drawn

    Techniques Used: Binding Assay

    35) Product Images from "The Herpesvirus saimiri Small Nuclear RNAs Recruit AU-Rich Element-Binding Proteins but Do Not Alter Host AU-Rich Element-Containing mRNA Levels in Virally Transformed T Cells"

    Article Title: The Herpesvirus saimiri Small Nuclear RNAs Recruit AU-Rich Element-Binding Proteins but Do Not Alter Host AU-Rich Element-Containing mRNA Levels in Virally Transformed T Cells

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.24.10.4522-4533.2004

    HSUR 1 interacts with hnRNP D and HuR in vivo. Intact HVS-transformed 1670 cells were irradiated with 254-nm UV light to induce cross-links between RNA and protein interacting in vivo (lanes 3, 5, and 7). Cells treated identically, but not cross-linked, served as negative controls (lanes 2, 4, and 6). Cell extracts were prepared under strongly denaturing conditions and subjected to immunoprecipitation with antibody to hnRNP D (lanes 4 and 5), HuR (lanes 6 and 7), or Sm proteins (lanes 2 and 3). Following digestion with proteinase K, immunoprecipitated RNA was isolated from the pellets and resolved along with 5% of total RNA (lane 1) in a 8% polyacrylamide gel. Northern blotting detected the ARE-containing HSUR 1 or HSUR 4 as a control for ARE-binding specificity. Small amounts of nonspecific RNAs of 150, 160, and 200 nucleotides (nt) were observed in all immunoprecipitation reactions.
    Figure Legend Snippet: HSUR 1 interacts with hnRNP D and HuR in vivo. Intact HVS-transformed 1670 cells were irradiated with 254-nm UV light to induce cross-links between RNA and protein interacting in vivo (lanes 3, 5, and 7). Cells treated identically, but not cross-linked, served as negative controls (lanes 2, 4, and 6). Cell extracts were prepared under strongly denaturing conditions and subjected to immunoprecipitation with antibody to hnRNP D (lanes 4 and 5), HuR (lanes 6 and 7), or Sm proteins (lanes 2 and 3). Following digestion with proteinase K, immunoprecipitated RNA was isolated from the pellets and resolved along with 5% of total RNA (lane 1) in a 8% polyacrylamide gel. Northern blotting detected the ARE-containing HSUR 1 or HSUR 4 as a control for ARE-binding specificity. Small amounts of nonspecific RNAs of 150, 160, and 200 nucleotides (nt) were observed in all immunoprecipitation reactions.

    Techniques Used: In Vivo, Transformation Assay, Irradiation, Immunoprecipitation, Isolation, Northern Blot, Binding Assay

    36) Product Images from "ZNF280BY and ZNF280AY: autosome derived Y-chromosome gene families in Bovidae"

    Article Title: ZNF280BY and ZNF280AY: autosome derived Y-chromosome gene families in Bovidae

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-12-13

    Tissue localization of the bovine ZNF280BY transcript in adult testis . The sense and antisense RNA of ZNF280BY are expressed in adult testis. A . The ZNF280BY sense RNA is expressed widely and evenly across all cell types in the seminiferous tubules. B . The antisense RNA of ZNF280BY was only detected in spermatids. Sense (A) and antisense (B) RNA of ZNF280BY were detected by the corresponding DIG-labeled cRNA probes. C . The bovine Protamine gene was used as the positive control, and there is no antisense mRNA of Protamine detected in the bovine testis [ 34 ]. D . The Haematoxylin and Eosin (H E) staining was shown. Scale: bar = 200 μm.
    Figure Legend Snippet: Tissue localization of the bovine ZNF280BY transcript in adult testis . The sense and antisense RNA of ZNF280BY are expressed in adult testis. A . The ZNF280BY sense RNA is expressed widely and evenly across all cell types in the seminiferous tubules. B . The antisense RNA of ZNF280BY was only detected in spermatids. Sense (A) and antisense (B) RNA of ZNF280BY were detected by the corresponding DIG-labeled cRNA probes. C . The bovine Protamine gene was used as the positive control, and there is no antisense mRNA of Protamine detected in the bovine testis [ 34 ]. D . The Haematoxylin and Eosin (H E) staining was shown. Scale: bar = 200 μm.

    Techniques Used: Labeling, Positive Control, Staining

    37) Product Images from "Induced ectopic expression of HigB toxin in Mycobacterium tuberculosis results in growth inhibition, reduced abundance of a subset of mRNAs and cleavage of tmRNA"

    Article Title: Induced ectopic expression of HigB toxin in Mycobacterium tuberculosis results in growth inhibition, reduced abundance of a subset of mRNAs and cleavage of tmRNA

    Journal: Molecular Microbiology

    doi: 10.1111/mmi.12358

    HigB cleavage sites identified by 3′ RACE. A schematic showing the DNA sequence of ssrA . Amino acids encoded in the mRNA like region are shown. Putative cleavage sites found in the 300 bp and 100–150 bp PCR products are indicated as highlighted 3′ residues before the A tail. Nucleotides between which the cleavage must have occurred are in bold. The sequence of the gene-specific forward primer used is underlined. The 5′ and 3′ probes used for Northern blots are shaded in grey.
    Figure Legend Snippet: HigB cleavage sites identified by 3′ RACE. A schematic showing the DNA sequence of ssrA . Amino acids encoded in the mRNA like region are shown. Putative cleavage sites found in the 300 bp and 100–150 bp PCR products are indicated as highlighted 3′ residues before the A tail. Nucleotides between which the cleavage must have occurred are in bold. The sequence of the gene-specific forward primer used is underlined. The 5′ and 3′ probes used for Northern blots are shaded in grey.

    Techniques Used: Sequencing, Polymerase Chain Reaction, Northern Blot

    38) Product Images from "Depletion of the RNA-Binding Protein RBP33 Results in Increased Expression of Silenced RNA Polymerase II Transcripts in Trypanosoma brucei"

    Article Title: Depletion of the RNA-Binding Protein RBP33 Results in Increased Expression of Silenced RNA Polymerase II Transcripts in Trypanosoma brucei

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0107608

    RBP33 is an essential protein in trypanosomes. Cell lines were generated that expressed RBP33 -specific dsRNA in a tetracycline-inducible manner. The levels of mRNA (A) or protein (B) were analyzed in cells incubated with tetracycline for 48 h. Loading controls were the 7SL RNA [40] or the CSM protein [39] . (C) RNAi cell lines were grown in the absence (open squares) or presence (filled circles) of 1 µg/ml tetracycline to induce RNAi. Cultures were followed up to 8 days and diluted to 0.5×10 5 cells/ml (bloodstream) or 0.4×10 6 cells/ml (procyclic) every 2 days as required.
    Figure Legend Snippet: RBP33 is an essential protein in trypanosomes. Cell lines were generated that expressed RBP33 -specific dsRNA in a tetracycline-inducible manner. The levels of mRNA (A) or protein (B) were analyzed in cells incubated with tetracycline for 48 h. Loading controls were the 7SL RNA [40] or the CSM protein [39] . (C) RNAi cell lines were grown in the absence (open squares) or presence (filled circles) of 1 µg/ml tetracycline to induce RNAi. Cultures were followed up to 8 days and diluted to 0.5×10 5 cells/ml (bloodstream) or 0.4×10 6 cells/ml (procyclic) every 2 days as required.

    Techniques Used: Generated, Incubation

    Effect of RBP33 depletion on the levels of SSR-derived transcripts. (A) Artmemis visualization of RNAseq data corresponding to the SSR downstream gene Tb927.7.1930 and to the SSR downstream gene Tb927.10.5710. Probes used for Northern blot analysis are indicated. ‘Input’ refers to the RNA-seq data obtained from total RNA before immunoprecipitation, whereas ‘RBP33-bound’ refers to the immunoprecipitated RNA using RBP33 specific antibodies. Annotated splice-acceptor and polyadenylation sites [13] are indicated as filled or empty circles, respectively. The splice-acceptor site for Tb927.10.5720 has been mapped in this study, and lies within the downstream open-reading frame. (B) Northern hybridizations using radiolabeled double-stranded DNA probes as indicated in panel A. An RNAi cell line expressing dsRNA against the essential RNA-binding protein DRBD3 [11] was used as a control. Total RNA was obtained from parasites grown in the absence (−) or the presence (+) of tetracycline for 48 h, transferred to nylon membranes and hybridized with specific probes. The 7SL RNA was used as a loading control. The arrow indicates the position of the 1.0 kb transcript detected with probe B. Representative blots are shown.
    Figure Legend Snippet: Effect of RBP33 depletion on the levels of SSR-derived transcripts. (A) Artmemis visualization of RNAseq data corresponding to the SSR downstream gene Tb927.7.1930 and to the SSR downstream gene Tb927.10.5710. Probes used for Northern blot analysis are indicated. ‘Input’ refers to the RNA-seq data obtained from total RNA before immunoprecipitation, whereas ‘RBP33-bound’ refers to the immunoprecipitated RNA using RBP33 specific antibodies. Annotated splice-acceptor and polyadenylation sites [13] are indicated as filled or empty circles, respectively. The splice-acceptor site for Tb927.10.5720 has been mapped in this study, and lies within the downstream open-reading frame. (B) Northern hybridizations using radiolabeled double-stranded DNA probes as indicated in panel A. An RNAi cell line expressing dsRNA against the essential RNA-binding protein DRBD3 [11] was used as a control. Total RNA was obtained from parasites grown in the absence (−) or the presence (+) of tetracycline for 48 h, transferred to nylon membranes and hybridized with specific probes. The 7SL RNA was used as a loading control. The arrow indicates the position of the 1.0 kb transcript detected with probe B. Representative blots are shown.

    Techniques Used: Derivative Assay, Northern Blot, RNA Sequencing Assay, Immunoprecipitation, Expressing, RNA Binding Assay

    39) Product Images from "The regulatory role of DR4 in a spontaneous diabetes DQ8 transgenic model"

    Article Title: The regulatory role of DR4 in a spontaneous diabetes DQ8 transgenic model

    Journal: Journal of Clinical Investigation

    doi:

    Immunohistochemistry staining of pancreatic sections of diabetic mice, with the diabetic DQ8 + /mII – /RIP-B7 mouse in the upper panel, diabetic DR4 + /mII – /RIP-B7 mouse in the middle panel, and diabetic DQ8 + DR4 + /mII – /RIP-B7 mouse in the lower panel. Islet infiltrates were stained for CD4 +  T cells (left), CD8 +  T cells (middle), and B220 +  B cells (right).
    Figure Legend Snippet: Immunohistochemistry staining of pancreatic sections of diabetic mice, with the diabetic DQ8 + /mII – /RIP-B7 mouse in the upper panel, diabetic DR4 + /mII – /RIP-B7 mouse in the middle panel, and diabetic DQ8 + DR4 + /mII – /RIP-B7 mouse in the lower panel. Islet infiltrates were stained for CD4 + T cells (left), CD8 + T cells (middle), and B220 + B cells (right).

    Techniques Used: Immunohistochemistry, Staining, Mouse Assay

    ( a ) Expression of HLA transgenes and the selection of CD4 +  T cells in BM chimeras. In the top panel, lymphocytes isolated from the spleens of three types of chimeras (as indicated) were stained with the B-cell marker B220 (PE) in combination with either anti–HLA-DQ (FITC) or DR (FITC). In the lower panel, the same splenocytes were stained with anti-CD4 (PE) and anti-CD8 (FITC). ( b ) Immunohistochemistry staining of pancreatic sections of BM chimeras. In the upper panel are DQ8 + /mII – /RIP-B7 BM chimera. DR4 + /mII – /RIP-B7 BM chimera are in the middle panel. The lower panel shows DQ8 + DR4 + /mII – /RIP-B7 BM chimera. Islet infiltrates were stained for CD4 +  T cells (left), CD8 +  T cells (middle), and B220 +  B cells (right).
    Figure Legend Snippet: ( a ) Expression of HLA transgenes and the selection of CD4 + T cells in BM chimeras. In the top panel, lymphocytes isolated from the spleens of three types of chimeras (as indicated) were stained with the B-cell marker B220 (PE) in combination with either anti–HLA-DQ (FITC) or DR (FITC). In the lower panel, the same splenocytes were stained with anti-CD4 (PE) and anti-CD8 (FITC). ( b ) Immunohistochemistry staining of pancreatic sections of BM chimeras. In the upper panel are DQ8 + /mII – /RIP-B7 BM chimera. DR4 + /mII – /RIP-B7 BM chimera are in the middle panel. The lower panel shows DQ8 + DR4 + /mII – /RIP-B7 BM chimera. Islet infiltrates were stained for CD4 + T cells (left), CD8 + T cells (middle), and B220 + B cells (right).

    Techniques Used: Expressing, Selection, Isolation, Staining, Marker, Immunohistochemistry

    Cytokine production by CD4 +  T cells after GAD ( a ) and anti-CD3 ( b ) stimulation. Purified CD4 +  cells (2 × 10 6 /ml) from spleens of nondiabetic mice, as indicated, were cultured with GAD protein or anti-CD3 (2C11 supernatant) in the presence of irradiated splenocytes for 72 and 48 hours, respectively. The culture supernatants were then tested for the production of IFN-γ and IL-4 using ELISA.
    Figure Legend Snippet: Cytokine production by CD4 + T cells after GAD ( a ) and anti-CD3 ( b ) stimulation. Purified CD4 + cells (2 × 10 6 /ml) from spleens of nondiabetic mice, as indicated, were cultured with GAD protein or anti-CD3 (2C11 supernatant) in the presence of irradiated splenocytes for 72 and 48 hours, respectively. The culture supernatants were then tested for the production of IFN-γ and IL-4 using ELISA.

    Techniques Used: Purification, Mouse Assay, Cell Culture, Irradiation, Enzyme-linked Immunosorbent Assay

    Expression of HLA transgenes and the selection of CD4 +  T cells. In the upper panel, lymphocytes isolated from peripheral blood in three types of mice (as indicated) were stained with the B-cell marker B220 (phycoerythrin; PE) in combination with either anti–HLA-DQ (FITC) or DR (FITC). In the middle panel, thymocytes were isolated from mice at 5–7 weeks of age and stained with anti-CD4 (PE;  y  axis) and anti-CD8 (FITC;  x  axis). Gated CD4 single-positive thymocytes were 5.2%, 5%, and 9.8% of total thymocytes analyzed in DQ8 + /mII – /RIP-B7, DR4 + /mII – /RIP-B7, and DQ8 + DR4 + /mII – /RIP-B7 mice, respectively. In the bottom panel, splenocytes (after removing erythrocytes) were stained with anti-CD4 (PE) and anti-CD8 (FITC). Gated CD4 single-positive splenocytes were 11.4%, 11.8%, and 19.5% of total splenocytes analyzed in DQ8 + /mII – /RIP-B7, DR4 + /mII – /RIP-B7, and DQ8 + DR4 + /mII – /RIP-B7 mice, respectively.
    Figure Legend Snippet: Expression of HLA transgenes and the selection of CD4 + T cells. In the upper panel, lymphocytes isolated from peripheral blood in three types of mice (as indicated) were stained with the B-cell marker B220 (phycoerythrin; PE) in combination with either anti–HLA-DQ (FITC) or DR (FITC). In the middle panel, thymocytes were isolated from mice at 5–7 weeks of age and stained with anti-CD4 (PE; y axis) and anti-CD8 (FITC; x axis). Gated CD4 single-positive thymocytes were 5.2%, 5%, and 9.8% of total thymocytes analyzed in DQ8 + /mII – /RIP-B7, DR4 + /mII – /RIP-B7, and DQ8 + DR4 + /mII – /RIP-B7 mice, respectively. In the bottom panel, splenocytes (after removing erythrocytes) were stained with anti-CD4 (PE) and anti-CD8 (FITC). Gated CD4 single-positive splenocytes were 11.4%, 11.8%, and 19.5% of total splenocytes analyzed in DQ8 + /mII – /RIP-B7, DR4 + /mII – /RIP-B7, and DQ8 + DR4 + /mII – /RIP-B7 mice, respectively.

    Techniques Used: Expressing, Selection, Isolation, Mouse Assay, Staining, Marker

    40) Product Images from "RNAi-mediated CCR5 Silencing by LFA-1-targeted Nanoparticles Prevents HIV Infection in BLT Mice"

    Article Title: RNAi-mediated CCR5 Silencing by LFA-1-targeted Nanoparticles Prevents HIV Infection in BLT Mice

    Journal: Molecular Therapy

    doi: 10.1038/mt.2009.271

    Systemic delivery of siCCR5 with LFA-1 I-tsNPs silences CCR5 expression in vivo . ( a ) siCCR5 uptake was monitored using RT-PCR in peripheral blood T cells (CD3 + ), B cells (CD19 + ), and monocytes (CD14 + ) in humanized mice. n.d., not
    Figure Legend Snippet: Systemic delivery of siCCR5 with LFA-1 I-tsNPs silences CCR5 expression in vivo . ( a ) siCCR5 uptake was monitored using RT-PCR in peripheral blood T cells (CD3 + ), B cells (CD19 + ), and monocytes (CD14 + ) in humanized mice. n.d., not

    Techniques Used: Expressing, In Vivo, Reverse Transcription Polymerase Chain Reaction, Mouse Assay

    Systemic delivery of siCCR5 with LFA-1 I-tsNPs protects BLT mice from HIV infection. ( a ) BLT mice were treated intravenously with either siLuc ( n = 3) or siCCR5 ( n = 3) complexed to LFA-1 I-tsNPs, infected with HIV BaL , and monitored for viral load and
    Figure Legend Snippet: Systemic delivery of siCCR5 with LFA-1 I-tsNPs protects BLT mice from HIV infection. ( a ) BLT mice were treated intravenously with either siLuc ( n = 3) or siCCR5 ( n = 3) complexed to LFA-1 I-tsNPs, infected with HIV BaL , and monitored for viral load and

    Techniques Used: Mouse Assay, Infection

    siRNA delivery to resting and activated T cells using nanoparticles targeted to LFA-1. ( a ) The size and zeta potential of carrier nanoparticles. ( b ) Binding of LFA-1 I-tsNPs to activated (blue) and naive (red) human primary lymphocytes. ( c ) CD4 silencing
    Figure Legend Snippet: siRNA delivery to resting and activated T cells using nanoparticles targeted to LFA-1. ( a ) The size and zeta potential of carrier nanoparticles. ( b ) Binding of LFA-1 I-tsNPs to activated (blue) and naive (red) human primary lymphocytes. ( c ) CD4 silencing

    Techniques Used: Binding Assay

    41) Product Images from "The mRNA cap methyltransferase gene TbCMT1 is not essential in vitro but is a virulence factor in vivo for bloodstream form Trypanosoma brucei"

    Article Title: The mRNA cap methyltransferase gene TbCMT1 is not essential in vitro but is a virulence factor in vivo for bloodstream form Trypanosoma brucei

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0201263

    TbCMT1 is not required for T . brucei Lister 427 BSF proliferation in cell culture. (A) Total RNA was purified from TbCMT1 conditional null cells cultured with (+tet) and without tetracycline for 24, 48 and 72 h. TbCMT1 expression was analysed by qRT-PCR normalised to telomerase reverse transcriptase ( TERT ). The delta Ct values (dCt) for nine measurements for each condition are visualised as a swarm plot to show all observations along with representations of the underlying distributions. (B) Cumulative cell counts of triplicate TbCMT1 conditional null mutant cell cultures grown with (plus) and without (minus) tetracycline. (C) Cumulative cell counts of triplicate wild type (WT) and TbCMT1 null cells cultured in parallel. For the data in panels B and C, the cell counts of three biological replicates are reported after 2, 4, 6, 8 and 10 days. The cultures were counted and diluted to 10 5 cells/ml every two days in (B) and to 10 4 cells/ml every two days in (C). The cell counts are reported as the log10 value of the cumulative number of parasites per ml of cell culture allowing for the aforementioned dilution factors.
    Figure Legend Snippet: TbCMT1 is not required for T . brucei Lister 427 BSF proliferation in cell culture. (A) Total RNA was purified from TbCMT1 conditional null cells cultured with (+tet) and without tetracycline for 24, 48 and 72 h. TbCMT1 expression was analysed by qRT-PCR normalised to telomerase reverse transcriptase ( TERT ). The delta Ct values (dCt) for nine measurements for each condition are visualised as a swarm plot to show all observations along with representations of the underlying distributions. (B) Cumulative cell counts of triplicate TbCMT1 conditional null mutant cell cultures grown with (plus) and without (minus) tetracycline. (C) Cumulative cell counts of triplicate wild type (WT) and TbCMT1 null cells cultured in parallel. For the data in panels B and C, the cell counts of three biological replicates are reported after 2, 4, 6, 8 and 10 days. The cultures were counted and diluted to 10 5 cells/ml every two days in (B) and to 10 4 cells/ml every two days in (C). The cell counts are reported as the log10 value of the cumulative number of parasites per ml of cell culture allowing for the aforementioned dilution factors.

    Techniques Used: Cell Culture, Purification, Expressing, Quantitative RT-PCR, Mutagenesis

    42) Product Images from "The exosome regulates circadian gene expression in a posttranscriptional negative feedback loop"

    Article Title: The exosome regulates circadian gene expression in a posttranscriptional negative feedback loop

    Journal: Cell

    doi: 10.1016/j.cell.2009.06.043

    FRH regulates the frq RNA decay by the exosome. (A) Northern blot analysis showing the decay of frq RNA after the addition of thiolutin. Due to high levels of frq RNA after down-regulation of FRH, a shorter exposure of the Northern blots for the QA treated samples was used. The densitometric analysis of the results from three independent experiments are shown in the bottom. (B) Northern blot analysis showing the results of the RNAse H assay for frq RNA. RNA samples from the wild-type and ds frh strains were used. Due to high levels of frq RNA in the ds frh samples, less RNA for the ds frh samples was applied to bring frq signals to comparable levels for both sets of the samples. (C) Western blot analysis showing the results of immunoprecipitation assay using the FRH antibody. Immunoprecipitation using the FRH pre-immune (PI) serum was used as the control. Myc-RRP44, Myc-RRP6 and Myc-QIP (the negative control) strains were used.
    Figure Legend Snippet: FRH regulates the frq RNA decay by the exosome. (A) Northern blot analysis showing the decay of frq RNA after the addition of thiolutin. Due to high levels of frq RNA after down-regulation of FRH, a shorter exposure of the Northern blots for the QA treated samples was used. The densitometric analysis of the results from three independent experiments are shown in the bottom. (B) Northern blot analysis showing the results of the RNAse H assay for frq RNA. RNA samples from the wild-type and ds frh strains were used. Due to high levels of frq RNA in the ds frh samples, less RNA for the ds frh samples was applied to bring frq signals to comparable levels for both sets of the samples. (C) Western blot analysis showing the results of immunoprecipitation assay using the FRH antibody. Immunoprecipitation using the FRH pre-immune (PI) serum was used as the control. Myc-RRP44, Myc-RRP6 and Myc-QIP (the negative control) strains were used.

    Techniques Used: Northern Blot, Rnase H Assay, Western Blot, Immunoprecipitation, Negative Control

    43) Product Images from "The roles of TRIO and F-actin-binding protein in glioblastoma cells"

    Article Title: The roles of TRIO and F-actin-binding protein in glioblastoma cells

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2018.8458

    Relative changes of TrioBP transcripts from GBM cells in standard RNA-seq data. Total RNA were isolated from two GBM cell lines (U87-MG and U251-MG) and normal brain tissue. These samples were further analyzed by the standard RNA deep sequencing (RNA-seq) as described in the material and methods. RNA-seq read density for TrioBP transcripts was plotted with the relative RNA-seq read coverage (counts). Fragments per kilobase of exon per million fragments mapped (FPKM) were calculated to compare the expression level of TrioBP mRNA variants in each sample.
    Figure Legend Snippet: Relative changes of TrioBP transcripts from GBM cells in standard RNA-seq data. Total RNA were isolated from two GBM cell lines (U87-MG and U251-MG) and normal brain tissue. These samples were further analyzed by the standard RNA deep sequencing (RNA-seq) as described in the material and methods. RNA-seq read density for TrioBP transcripts was plotted with the relative RNA-seq read coverage (counts). Fragments per kilobase of exon per million fragments mapped (FPKM) were calculated to compare the expression level of TrioBP mRNA variants in each sample.

    Techniques Used: RNA Sequencing Assay, Isolation, Sequencing, Expressing

    44) Product Images from "Induced ectopic expression of HigB toxin in Mycobacterium tuberculosis results in growth inhibition, reduced abundance of a subset of mRNAs and cleavage of tmRNA"

    Article Title: Induced ectopic expression of HigB toxin in Mycobacterium tuberculosis results in growth inhibition, reduced abundance of a subset of mRNAs and cleavage of tmRNA

    Journal: Molecular Microbiology

    doi: 10.1111/mmi.12358

    HigB expression affects tmRNA in M. smegmatis and E. coli .A. DNA alignment of ssrA from M. tuberculosis and E. coli . Conserved residues are highlighted by an asterisk (*).B. Northern blots probing for tmRNA (B) and 5 S (C). RNA was extracted from M. smegmatis (left panel) and E. coli (right panel) HigB expression strains grown with or without inducer. Strains were grown to mid-exponential phase and inducer was added; ATc for M. smegmatis and l -arabinose for E. coli , for 7.5 h and 2 h respectively.
    Figure Legend Snippet: HigB expression affects tmRNA in M. smegmatis and E. coli .A. DNA alignment of ssrA from M. tuberculosis and E. coli . Conserved residues are highlighted by an asterisk (*).B. Northern blots probing for tmRNA (B) and 5 S (C). RNA was extracted from M. smegmatis (left panel) and E. coli (right panel) HigB expression strains grown with or without inducer. Strains were grown to mid-exponential phase and inducer was added; ATc for M. smegmatis and l -arabinose for E. coli , for 7.5 h and 2 h respectively.

    Techniques Used: Expressing, Northern Blot

    45) Product Images from "STRL33, A Novel Chemokine Receptor-like Protein, Functions as a Fusion Cofactor for Both Macrophage-tropic and T Cell Line-tropic HIV-1"

    Article Title: STRL33, A Novel Chemokine Receptor-like Protein, Functions as a Fusion Cofactor for Both Macrophage-tropic and T Cell Line-tropic HIV-1

    Journal: The Journal of Experimental Medicine

    doi:

    Expression of STRL33 and other GPCR genes. ( A ) The expression of STRL33 , genes for known chemokine receptors, and genes for selected orphan GPCRs in leukocytes. 15 μg of total RNA were electrophoresed on 1.2% agarose–formaldehyde gels, transferred to nitrocellulose membranes, and hybridized with the probes indicated on the left. A total of six membranes were used for hybridizations, and adequate removal of signal was documented before repeat probings. Film exposure times ranged from overnight for the IL-8RA and IL-8RB blots to 13 d for the CXCR4 blot. Probings were done using an oligonucleotide complementary to 18S rRNA in order to demonstrate amounts of RNA loaded per lane and a representative blot is shown. ( B ) The expression of STRL33 in activated PBL. 25 μg of total RNA from TIL, and freshly isolated and activated PBL were analyzed as in A and hybridized with a 32 P-labeled STRL33 ORF probe and a probe for 18S rRNA.
    Figure Legend Snippet: Expression of STRL33 and other GPCR genes. ( A ) The expression of STRL33 , genes for known chemokine receptors, and genes for selected orphan GPCRs in leukocytes. 15 μg of total RNA were electrophoresed on 1.2% agarose–formaldehyde gels, transferred to nitrocellulose membranes, and hybridized with the probes indicated on the left. A total of six membranes were used for hybridizations, and adequate removal of signal was documented before repeat probings. Film exposure times ranged from overnight for the IL-8RA and IL-8RB blots to 13 d for the CXCR4 blot. Probings were done using an oligonucleotide complementary to 18S rRNA in order to demonstrate amounts of RNA loaded per lane and a representative blot is shown. ( B ) The expression of STRL33 in activated PBL. 25 μg of total RNA from TIL, and freshly isolated and activated PBL were analyzed as in A and hybridized with a 32 P-labeled STRL33 ORF probe and a probe for 18S rRNA.

    Techniques Used: Expressing, Isolation, Labeling

    46) Product Images from "Polycomb Protein OsFIE2 Affects Plant Height and Grain Yield in Rice"

    Article Title: Polycomb Protein OsFIE2 Affects Plant Height and Grain Yield in Rice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0164748

    RNA-seq-based and qPCR analysis of differentially expressed genes. A, GO enrichment analysis of differentially expressed genes (DEGs). B, Heatmap showing the expression levels of DEGs in osfie2-1 according to RNA-seq. C, Expression level of homeobox OsMADS-BOX genes and Oskn3 in wild-type and osfie2-1 . D, Expression level of phytohormone-related genes. E-I, Phytohormones content in wild-type and osfie2-1 . Data are given as mean ± SD of three biological replicates. Student’s t-test was used to generate the P values; * and ** indicate P
    Figure Legend Snippet: RNA-seq-based and qPCR analysis of differentially expressed genes. A, GO enrichment analysis of differentially expressed genes (DEGs). B, Heatmap showing the expression levels of DEGs in osfie2-1 according to RNA-seq. C, Expression level of homeobox OsMADS-BOX genes and Oskn3 in wild-type and osfie2-1 . D, Expression level of phytohormone-related genes. E-I, Phytohormones content in wild-type and osfie2-1 . Data are given as mean ± SD of three biological replicates. Student’s t-test was used to generate the P values; * and ** indicate P

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

    47) Product Images from "Molecular Cloning Reveals that the p160 Myb-Binding Protein Is a Novel, Predominantly Nucleolar Protein Which May Play a Role in Transactivation by Myb"

    Article Title: Molecular Cloning Reveals that the p160 Myb-Binding Protein Is a Novel, Predominantly Nucleolar Protein Which May Play a Role in Transactivation by Myb

    Journal: Molecular and Cellular Biology

    doi:

    Northern blot analysis of p160 mRNA expression with the p160 cDNA probe. In the upper panels, 2 μg of poly(A) + RNA from the indicated cell lines was loaded in each lane (MTHC, myb transformed hemopoietic cells) (A) or 30 μg of total RNA from the indicated mouse tissues (or FDC-P1 cells) was loaded in each lane (B). The size of the p160 mRNA was determined by comparison with RNA markers (not shown). In the lower panels, the Northern blots shown in the upper panels were stripped and reprobed with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA as a control for mRNA loading; only the relevant area of each blot is shown.
    Figure Legend Snippet: Northern blot analysis of p160 mRNA expression with the p160 cDNA probe. In the upper panels, 2 μg of poly(A) + RNA from the indicated cell lines was loaded in each lane (MTHC, myb transformed hemopoietic cells) (A) or 30 μg of total RNA from the indicated mouse tissues (or FDC-P1 cells) was loaded in each lane (B). The size of the p160 mRNA was determined by comparison with RNA markers (not shown). In the lower panels, the Northern blots shown in the upper panels were stripped and reprobed with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA as a control for mRNA loading; only the relevant area of each blot is shown.

    Techniques Used: Northern Blot, Expressing, Transformation Assay

    48) Product Images from "Distinct chromatin functional states correlate with HIV latency reactivation in infected primary CD4+ T cells"

    Article Title: Distinct chromatin functional states correlate with HIV latency reactivation in infected primary CD4+ T cells

    Journal: eLife

    doi: 10.7554/eLife.34655

    LRAs efficacy in patient samples is predicted by activity in HIV GKO latently infected cells. ( A ) Intracellular HIV-1 mRNA levels in rCD4s, obtained from infected individuals and treated ex vivo with a single LRA or a combination of two LRAs for 24 hr in presence of raltegravir, presented as fold induction relative to DMSO control. (n = 4, mean +SEM) ( Figure 2—source data 1 ). ( B ) Intracellular HIV-1 mRNA levels in HIV GKO latently infected CD4 + T-cells, and treated with a single LRA or a combination of two LRAs for 6 hr in presence of raltegravir, presented as fold induction relative to DMSO control. (n = 3 (different donors), mean +SEM, paired t-test) ( Figure 2—source data 1 ). ( C ) Correlation between intracellular HIV-1 mRNA levels quantified in either 6 hr stimulated HIV GKO latently infected CD4 + T-cells from different donors, or 24 hr stimulated rCD4s from HIV infected patients, with a single LRA or a combination of two LRAs in presence of raltegravir. 10.7554/eLife.34655.006 Intracellular HIV-1 mRNA levels in rCD4s, obtained from infected individuals, or in HIV GKO latently infected CD4 + T-cells. The experiment is detailed in the main text and Figure 2 legend.
    Figure Legend Snippet: LRAs efficacy in patient samples is predicted by activity in HIV GKO latently infected cells. ( A ) Intracellular HIV-1 mRNA levels in rCD4s, obtained from infected individuals and treated ex vivo with a single LRA or a combination of two LRAs for 24 hr in presence of raltegravir, presented as fold induction relative to DMSO control. (n = 4, mean +SEM) ( Figure 2—source data 1 ). ( B ) Intracellular HIV-1 mRNA levels in HIV GKO latently infected CD4 + T-cells, and treated with a single LRA or a combination of two LRAs for 6 hr in presence of raltegravir, presented as fold induction relative to DMSO control. (n = 3 (different donors), mean +SEM, paired t-test) ( Figure 2—source data 1 ). ( C ) Correlation between intracellular HIV-1 mRNA levels quantified in either 6 hr stimulated HIV GKO latently infected CD4 + T-cells from different donors, or 24 hr stimulated rCD4s from HIV infected patients, with a single LRA or a combination of two LRAs in presence of raltegravir. 10.7554/eLife.34655.006 Intracellular HIV-1 mRNA levels in rCD4s, obtained from infected individuals, or in HIV GKO latently infected CD4 + T-cells. The experiment is detailed in the main text and Figure 2 legend.

    Techniques Used: Activity Assay, Infection, Ex Vivo

    (1)Intracellular HIV-1 mRNA levels in rCD4s, obtained from infected individuals and treated ex vivo with a single LRA or a combination of two LRAs for 24 hours, presented as fold induction relative to DMSO control.
    Figure Legend Snippet: (1)Intracellular HIV-1 mRNA levels in rCD4s, obtained from infected individuals and treated ex vivo with a single LRA or a combination of two LRAs for 24 hours, presented as fold induction relative to DMSO control.

    Techniques Used: Infection, Ex Vivo

    49) Product Images from "A Combinational Strategy upon RNA Sequencing and Peptidomics Unravels a Set of Novel Toxin Peptides in Scorpion Mesobuthus martensii"

    Article Title: A Combinational Strategy upon RNA Sequencing and Peptidomics Unravels a Set of Novel Toxin Peptides in Scorpion Mesobuthus martensii

    Journal: Toxins

    doi: 10.3390/toxins8100286

    Alignment of potassium channel toxins from M. martensii . ( A ) Twelve potassium channel toxins aligned based on sequence similarity and all toxins had six cysteines; ( B ) Other identified potassium channel toxins.
    Figure Legend Snippet: Alignment of potassium channel toxins from M. martensii . ( A ) Twelve potassium channel toxins aligned based on sequence similarity and all toxins had six cysteines; ( B ) Other identified potassium channel toxins.

    Techniques Used: Sequencing

    Alignment of sodium channel toxins from M. martensii . All sodium channel toxins were aligned by similarity. Sequences of sodium channel toxins belong to family 1 ( A ) and family 2 ( B ); ( C ) Three other sodium channel toxins identified from scorpion venom with low similarity were found; ( D ) Sequence alignment of LqhIT2-1 and LqhIT2.
    Figure Legend Snippet: Alignment of sodium channel toxins from M. martensii . All sodium channel toxins were aligned by similarity. Sequences of sodium channel toxins belong to family 1 ( A ) and family 2 ( B ); ( C ) Three other sodium channel toxins identified from scorpion venom with low similarity were found; ( D ) Sequence alignment of LqhIT2-1 and LqhIT2.

    Techniques Used: Sequencing

    50) Product Images from "Regulation of p110? PI 3-Kinase Gene Expression"

    Article Title: Regulation of p110? PI 3-Kinase Gene Expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0005145

    Equal p110δ mRNA stability in leukocytes and non-leukocytes. The indicated cell lines were treated with Actinomycin D (4 µg/ml), an inhibitor of de novo RNA synthesis, for the indicated time points followed by quantification of either p110δ mRNA (A) or p110δ protein (B). p110δ mRNA was quantified by real time RT-PCR, using normalisation for 18S RNA. p110δ mRNA levels are presented in a semi-log plot.
    Figure Legend Snippet: Equal p110δ mRNA stability in leukocytes and non-leukocytes. The indicated cell lines were treated with Actinomycin D (4 µg/ml), an inhibitor of de novo RNA synthesis, for the indicated time points followed by quantification of either p110δ mRNA (A) or p110δ protein (B). p110δ mRNA was quantified by real time RT-PCR, using normalisation for 18S RNA. p110δ mRNA levels are presented in a semi-log plot.

    Techniques Used: Quantitative RT-PCR

    51) Product Images from "Functional and Structural Analysis of Maize Hsp101 IRES"

    Article Title: Functional and Structural Analysis of Maize Hsp101 IRES

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0107459

    Identification of proteins associated to maize hsp101 IRES. Purification of IRES-protein complexes using RRL translation system (a), or Lb-treated RRL translation system (b) with RNAs indicated at the top. Equal amounts of samples containing the proteins that coprecipitated with the indicated RNAs were loaded on SDS-PAGE and detected by silver staining. Red arrows depict two bands specifically copurifying with 101+1 sense RNA using the Lb-treated RRL. c) Sequence of the 75 kDa protein that binds to maize hsp101 IRES. The 75 KDa band (indicated by an arrow in lane 2 of panel b) was sequenced and identified as the molecular chaperone HSP90. The results yielded 4 unique peptides (shaded in yellow), 4 unique spectra, 5 total spectra, 54/565 amino acids (10% coverage).
    Figure Legend Snippet: Identification of proteins associated to maize hsp101 IRES. Purification of IRES-protein complexes using RRL translation system (a), or Lb-treated RRL translation system (b) with RNAs indicated at the top. Equal amounts of samples containing the proteins that coprecipitated with the indicated RNAs were loaded on SDS-PAGE and detected by silver staining. Red arrows depict two bands specifically copurifying with 101+1 sense RNA using the Lb-treated RRL. c) Sequence of the 75 kDa protein that binds to maize hsp101 IRES. The 75 KDa band (indicated by an arrow in lane 2 of panel b) was sequenced and identified as the molecular chaperone HSP90. The results yielded 4 unique peptides (shaded in yellow), 4 unique spectra, 5 total spectra, 54/565 amino acids (10% coverage).

    Techniques Used: Purification, SDS Page, Silver Staining, Sequencing

    Effect of HSP90 protein on hsp101+1 translation initiation. Equal amounts of in vitro synthesized RNAs 101+1 were used to program translation using RRL in the presence of Radicicol (lane 3), anti-HSP90 (lane 4), HSP90 protein (lane 5), or DMSO (lane 6). Proteins were resolved by SDS–PAGE and visualized by autoradiography of the dry gel. Synthesis of luciferase driven by the indicated IRES elements in the presence of the Lb protease (cap-independent).
    Figure Legend Snippet: Effect of HSP90 protein on hsp101+1 translation initiation. Equal amounts of in vitro synthesized RNAs 101+1 were used to program translation using RRL in the presence of Radicicol (lane 3), anti-HSP90 (lane 4), HSP90 protein (lane 5), or DMSO (lane 6). Proteins were resolved by SDS–PAGE and visualized by autoradiography of the dry gel. Synthesis of luciferase driven by the indicated IRES elements in the presence of the Lb protease (cap-independent).

    Techniques Used: In Vitro, Synthesized, SDS Page, Autoradiography, Luciferase

    Functional analysis of the maize hsp101 IRES. a) Maize Hsp101 5′UTR sequence. Arrows indicate the first nucleotide of the fragments used to identify the functional IRES region. b) Schematic representation of the bicistronic constructs pBIC-5 plasmid was used as positive control. Hsp101 fragments (sense orientation) were inserted between the chloramphenicol acetyl transferase (CAT) and luciferase (Luc) reporter genes. c) Translation efficiency of the mutant IRES elements in RRL. Equal amounts of in vitro synthesized RNAs pBIC-5, 101+1, 101Δ17 or 101Δ50 were used to program translation using Lb-untreated (-) RRL or Lb-treated (+) RRL. Proteins were resolved by SDS–PAGE and visualized by autoradiography of dry gels. Synthesis of luciferase driven by the indicated IRES elements in the presence or absence of the Lb protease is shown on the insert. The histogram shows the intensity of luciferase translation efficiency driven by the indicated RNAs in the presence of Lb (cap-independent).
    Figure Legend Snippet: Functional analysis of the maize hsp101 IRES. a) Maize Hsp101 5′UTR sequence. Arrows indicate the first nucleotide of the fragments used to identify the functional IRES region. b) Schematic representation of the bicistronic constructs pBIC-5 plasmid was used as positive control. Hsp101 fragments (sense orientation) were inserted between the chloramphenicol acetyl transferase (CAT) and luciferase (Luc) reporter genes. c) Translation efficiency of the mutant IRES elements in RRL. Equal amounts of in vitro synthesized RNAs pBIC-5, 101+1, 101Δ17 or 101Δ50 were used to program translation using Lb-untreated (-) RRL or Lb-treated (+) RRL. Proteins were resolved by SDS–PAGE and visualized by autoradiography of dry gels. Synthesis of luciferase driven by the indicated IRES elements in the presence or absence of the Lb protease is shown on the insert. The histogram shows the intensity of luciferase translation efficiency driven by the indicated RNAs in the presence of Lb (cap-independent).

    Techniques Used: Functional Assay, Sequencing, Construct, Plasmid Preparation, Positive Control, Chloramphenicol Acetyltransferase Assay, Luciferase, Mutagenesis, In Vitro, Synthesized, SDS Page, Autoradiography

    SHAPE structural analysis of maize hsp101 IRES. a) Primer extension analysis of 101+1 RNA. The first four lanes (UACG) show hsp101+1 sequence, obtained with the 5′end labeled primer. Lanes 5 and 6 show the primer extension products obtained with 101+1 RNA untreated (−) or treated (+) with NMIA, respectively b) SHAPE reactivity of 101+1 RNA. Reactivity was normalized relative to the full-length product (set to 100%). Blue bars represent values
    Figure Legend Snippet: SHAPE structural analysis of maize hsp101 IRES. a) Primer extension analysis of 101+1 RNA. The first four lanes (UACG) show hsp101+1 sequence, obtained with the 5′end labeled primer. Lanes 5 and 6 show the primer extension products obtained with 101+1 RNA untreated (−) or treated (+) with NMIA, respectively b) SHAPE reactivity of 101+1 RNA. Reactivity was normalized relative to the full-length product (set to 100%). Blue bars represent values

    Techniques Used: Sequencing, Labeling

    52) Product Images from "Molecular Cloning Reveals that the p160 Myb-Binding Protein Is a Novel, Predominantly Nucleolar Protein Which May Play a Role in Transactivation by Myb"

    Article Title: Molecular Cloning Reveals that the p160 Myb-Binding Protein Is a Novel, Predominantly Nucleolar Protein Which May Play a Role in Transactivation by Myb

    Journal: Molecular and Cellular Biology

    doi:

    Northern blot analysis of p160 mRNA expression with the p160 cDNA probe. In the upper panels, 2 μg of poly(A) + RNA from the indicated cell lines was loaded in each lane (MTHC, myb transformed hemopoietic cells) (A) or 30 μg of total RNA from the indicated mouse tissues (or FDC-P1 cells) was loaded in each lane (B). The size of the p160 mRNA was determined by comparison with RNA markers (not shown). In the lower panels, the Northern blots shown in the upper panels were stripped and reprobed with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA as a control for mRNA loading; only the relevant area of each blot is shown.
    Figure Legend Snippet: Northern blot analysis of p160 mRNA expression with the p160 cDNA probe. In the upper panels, 2 μg of poly(A) + RNA from the indicated cell lines was loaded in each lane (MTHC, myb transformed hemopoietic cells) (A) or 30 μg of total RNA from the indicated mouse tissues (or FDC-P1 cells) was loaded in each lane (B). The size of the p160 mRNA was determined by comparison with RNA markers (not shown). In the lower panels, the Northern blots shown in the upper panels were stripped and reprobed with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA as a control for mRNA loading; only the relevant area of each blot is shown.

    Techniques Used: Northern Blot, Expressing, Transformation Assay

    Proteins detected by antisera raised to the N and C termini of p160. Cytoplasmic (Cyt) and nuclear (Nuc) extracts from FDC-P1 and NIH 3T3 cells, as indicated, were fractionated by SDS-PAGE and immunoblotted with antisera against the N-terminal (anti-160N) (A) or the C-terminal (anti-160C) (B) regions of p160. The positions of p160 and p67 are indicated by arrows, and those of size standards (in kilodaltons) are indicated at the left.
    Figure Legend Snippet: Proteins detected by antisera raised to the N and C termini of p160. Cytoplasmic (Cyt) and nuclear (Nuc) extracts from FDC-P1 and NIH 3T3 cells, as indicated, were fractionated by SDS-PAGE and immunoblotted with antisera against the N-terminal (anti-160N) (A) or the C-terminal (anti-160C) (B) regions of p160. The positions of p160 and p67 are indicated by arrows, and those of size standards (in kilodaltons) are indicated at the left.

    Techniques Used: SDS Page

    Proteolysis of p160 by cell extracts. In vitro-translated p160 labelled with [ 35 S]methionine was incubated with extracts from FDC-P1 or NIH 3T3 cells for the indicated times (in minutes), after which the products were analyzed by SDS-PAGE. The positions and molecular masses of size standards are indicated at the right, and the bands corresponding to p160 and the 67-kDa product (p67) are indicated at the left.
    Figure Legend Snippet: Proteolysis of p160 by cell extracts. In vitro-translated p160 labelled with [ 35 S]methionine was incubated with extracts from FDC-P1 or NIH 3T3 cells for the indicated times (in minutes), after which the products were analyzed by SDS-PAGE. The positions and molecular masses of size standards are indicated at the right, and the bands corresponding to p160 and the 67-kDa product (p67) are indicated at the left.

    Techniques Used: In Vitro, Incubation, SDS Page

    53) Product Images from "Pioglitazone, a PPAR? Agonist, Suppresses CYP19 Transcription: Evidence for Involvement of 15-Hydroxyprostaglandin Dehydrogenase and BRCA1"

    Article Title: Pioglitazone, a PPAR? Agonist, Suppresses CYP19 Transcription: Evidence for Involvement of 15-Hydroxyprostaglandin Dehydrogenase and BRCA1

    Journal: Cancer prevention research (Philadelphia, Pa.)

    doi: 10.1158/1940-6207.CAPR-12-0201

    Pioglitazone induces 15-PGDH and BRCA1, and suppresses aromatase levels in mouse mammary gland. Mice were fed control diet or control diet supplemented with 0.05% or 0.1% (w/w) pioglitazone for 2 weeks before being sacrificed. Levels of aromatase activity (A), aromatase mRNA (B), 15-PGDH mRNA (C), PGE 2 (D) 13,14-dihydro-15-keto PGE 2 (E), and BRCA1 mRNA (F) were measured in mammary glands. A, aromatase activity is expressed as femtomoles/μg protein/h. Total RNA was prepared and aromatase (B), 15-PGDH (C), and BRCA1 (F) mRNA levels were quantified by real-time PCR. D and E, PGE 2 and 13,14-dihydro-15-keto-PGE 2 levels were measured in mammary glands. The biomarker levels under each experimental condition ( n = 10) were summarized in terms of mean ± SD ■, P
    Figure Legend Snippet: Pioglitazone induces 15-PGDH and BRCA1, and suppresses aromatase levels in mouse mammary gland. Mice were fed control diet or control diet supplemented with 0.05% or 0.1% (w/w) pioglitazone for 2 weeks before being sacrificed. Levels of aromatase activity (A), aromatase mRNA (B), 15-PGDH mRNA (C), PGE 2 (D) 13,14-dihydro-15-keto PGE 2 (E), and BRCA1 mRNA (F) were measured in mammary glands. A, aromatase activity is expressed as femtomoles/μg protein/h. Total RNA was prepared and aromatase (B), 15-PGDH (C), and BRCA1 (F) mRNA levels were quantified by real-time PCR. D and E, PGE 2 and 13,14-dihydro-15-keto-PGE 2 levels were measured in mammary glands. The biomarker levels under each experimental condition ( n = 10) were summarized in terms of mean ± SD ■, P

    Techniques Used: Mouse Assay, Activity Assay, Real-time Polymerase Chain Reaction, Biomarker Assay

    Pioglitazone-mediated downregulation of Egr-1 and Snail induces 15-PGDH. A, cells were treated with indicated concentrations of pioglitazone for 12 hours. Total RNA was prepared and 15-PGDH mRNA was quantified by real-time PCR. Values were normalized to the levels of β-actin. In the inset, Western blotting was conducted and the blot was probed with antibodies to 15-PGDH and β-actin. B and C, cells were transfected with 2 μg of control siRNA (GFP) or 15-PGDH siRNA as indicated. 24 hours after transfection, cells were treated with vehicle or 10 μmol/L pioglitazone for an additional 24 hours. B, levels of PGE 2 in the cell culture medium were quantified by enzyme immunoassay. In the inset, Western blotting was conducted and the blot was probed with antibodies to 15-PGDH and β-actin. C, aromatase activity was measured. Enzyme activity is expressed as femtomoles/μg protein/min. D and E, cells were treated with indicated concentrations of pioglitazone for 8 and 10 hours, respectively. Cells were then lysed and 100 μg of cell lysate protein was subjected to Western blotting. The blots were probed with antibodies to Egr-1, Snail, and β-actin as indicated. F–H, cells were transfected with 2 μg of control (GFP) or Egr-1 siRNA. Western blotting was performed and blots were probed as indicated with antibodies to Egr-1, Snail, 15-PGDH and β-actin. I, ChIP assays were conducted. Cells were treated with vehicle (control) or 10 μmol/L pioglitazone for 10 hours. Chromatin fragments were immunoprecipitated with antibody against Snail and the 15-PGDH promoter was amplified by real-time PCR. DNA sequencing was carried out, and the PCR product was confirmed to be the 15-PGDH promoter. The 15-PGDH promoter was not detected when normal IgG was used or antibody was omitted from the immunoprecipitation step (data not shown). Mean ± SD are shown, n = 3. * , P
    Figure Legend Snippet: Pioglitazone-mediated downregulation of Egr-1 and Snail induces 15-PGDH. A, cells were treated with indicated concentrations of pioglitazone for 12 hours. Total RNA was prepared and 15-PGDH mRNA was quantified by real-time PCR. Values were normalized to the levels of β-actin. In the inset, Western blotting was conducted and the blot was probed with antibodies to 15-PGDH and β-actin. B and C, cells were transfected with 2 μg of control siRNA (GFP) or 15-PGDH siRNA as indicated. 24 hours after transfection, cells were treated with vehicle or 10 μmol/L pioglitazone for an additional 24 hours. B, levels of PGE 2 in the cell culture medium were quantified by enzyme immunoassay. In the inset, Western blotting was conducted and the blot was probed with antibodies to 15-PGDH and β-actin. C, aromatase activity was measured. Enzyme activity is expressed as femtomoles/μg protein/min. D and E, cells were treated with indicated concentrations of pioglitazone for 8 and 10 hours, respectively. Cells were then lysed and 100 μg of cell lysate protein was subjected to Western blotting. The blots were probed with antibodies to Egr-1, Snail, and β-actin as indicated. F–H, cells were transfected with 2 μg of control (GFP) or Egr-1 siRNA. Western blotting was performed and blots were probed as indicated with antibodies to Egr-1, Snail, 15-PGDH and β-actin. I, ChIP assays were conducted. Cells were treated with vehicle (control) or 10 μmol/L pioglitazone for 10 hours. Chromatin fragments were immunoprecipitated with antibody against Snail and the 15-PGDH promoter was amplified by real-time PCR. DNA sequencing was carried out, and the PCR product was confirmed to be the 15-PGDH promoter. The 15-PGDH promoter was not detected when normal IgG was used or antibody was omitted from the immunoprecipitation step (data not shown). Mean ± SD are shown, n = 3. * , P

    Techniques Used: Real-time Polymerase Chain Reaction, Western Blot, Transfection, Cell Culture, Enzyme-linked Immunosorbent Assay, Activity Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Amplification, DNA Sequencing, Polymerase Chain Reaction

    Pioglitazone induces BRCA1 in preadipocytes. A, cells were treated with indicated concentrations of pioglitazone for 24 hours. Subsequently, total RNA was isolated and BRCA1 mRNA was quantified by real-time PCR. Values were normalized to levels of β-actin. B, cells were transfected with 1.8 μg BRCA1 promoter-luciferase. C, ChIP assays were conducted. Chromatin fragments were immunoprecipitated with antibody against PPARγ and the BRCA1 promoter was amplified by PCR (top) or real-time PCR (bottom). DNA sequencing was carried out, and the PCR product was confirmed to be the BRCA1 promoter. The BRCA1 promoter was not detected when normal IgG was used or antibody was omitted from the immunoprecipitation step (data not shown). Mean ± SD are shown, n =3. * , P
    Figure Legend Snippet: Pioglitazone induces BRCA1 in preadipocytes. A, cells were treated with indicated concentrations of pioglitazone for 24 hours. Subsequently, total RNA was isolated and BRCA1 mRNA was quantified by real-time PCR. Values were normalized to levels of β-actin. B, cells were transfected with 1.8 μg BRCA1 promoter-luciferase. C, ChIP assays were conducted. Chromatin fragments were immunoprecipitated with antibody against PPARγ and the BRCA1 promoter was amplified by PCR (top) or real-time PCR (bottom). DNA sequencing was carried out, and the PCR product was confirmed to be the BRCA1 promoter. The BRCA1 promoter was not detected when normal IgG was used or antibody was omitted from the immunoprecipitation step (data not shown). Mean ± SD are shown, n =3. * , P

    Techniques Used: Isolation, Real-time Polymerase Chain Reaction, Transfection, Luciferase, Chromatin Immunoprecipitation, Immunoprecipitation, Amplification, Polymerase Chain Reaction, DNA Sequencing

    Pioglitazone inhibits aromatase expression in human preadipocytes. A, cells were transfected with 1.8 μg PPRE-luciferase and 0.2 μg pSVβgal. Following transfection, cells were treated with the indicated concentrations of pioglitazone for 24 hours. In A, D, and E, cells were lysed and luciferase activity was measured in cell lysates. Firefly luciferase activity was normalized to β-galactosidase activity. B, cells were treated with the indicated concentration of pioglitazone for 24 hours and then aromatase activity was assayed in cell lysates. Enzyme activity is expressed as femtomoles/μg protein/min. C, total RNA was prepared from cells that had been treated with pioglitazone for 24 hours. Levels of aromatase mRNA were quantified by real-time PCR. Values were normalized to levels of β-actin. D, cells were transfected with 1.8 μg CYP19 I.3/II promoter and 0.2 μg pSVβgal. Cells were then treated with indicated concentrations of pioglitazone for 24 hours. E, cells were transfected with 0.9 μg CYP19 I.3/II promoter and 0.2 μg pSVβgal. Cells also received 0.9 μg siRNA to GFP (control siRNA) or PPARγ. 24 hours after transfection, cells were treated with vehicle or 10 μmol/L pioglitazone. Aromatase promoter activity was measured 24 hours after treatment. In the inset, Western blotting was conducted on cell lysates that were treated with siRNA to GFP (control siRNA) or PPARγ. The blot was probed with antibodies to PPARγ and β-actin. F, cells were treated with indicated concentrations of PGE 2 . 24 hours later, aromatase activity was determined. G, cells were treated with indicated concentrations of pioglitazone for 16 hours. Subsequently, the concentration of PGE 2 in the cell culture medium was measured using enzyme immunoassay. In H and I, cells were treated with pioglitazone for 18 hours. Subsequently, cellular levels of cAMP (H) and PKA activity (I) were determined. A–I, mean ± SD are shown, n = 6. * , P
    Figure Legend Snippet: Pioglitazone inhibits aromatase expression in human preadipocytes. A, cells were transfected with 1.8 μg PPRE-luciferase and 0.2 μg pSVβgal. Following transfection, cells were treated with the indicated concentrations of pioglitazone for 24 hours. In A, D, and E, cells were lysed and luciferase activity was measured in cell lysates. Firefly luciferase activity was normalized to β-galactosidase activity. B, cells were treated with the indicated concentration of pioglitazone for 24 hours and then aromatase activity was assayed in cell lysates. Enzyme activity is expressed as femtomoles/μg protein/min. C, total RNA was prepared from cells that had been treated with pioglitazone for 24 hours. Levels of aromatase mRNA were quantified by real-time PCR. Values were normalized to levels of β-actin. D, cells were transfected with 1.8 μg CYP19 I.3/II promoter and 0.2 μg pSVβgal. Cells were then treated with indicated concentrations of pioglitazone for 24 hours. E, cells were transfected with 0.9 μg CYP19 I.3/II promoter and 0.2 μg pSVβgal. Cells also received 0.9 μg siRNA to GFP (control siRNA) or PPARγ. 24 hours after transfection, cells were treated with vehicle or 10 μmol/L pioglitazone. Aromatase promoter activity was measured 24 hours after treatment. In the inset, Western blotting was conducted on cell lysates that were treated with siRNA to GFP (control siRNA) or PPARγ. The blot was probed with antibodies to PPARγ and β-actin. F, cells were treated with indicated concentrations of PGE 2 . 24 hours later, aromatase activity was determined. G, cells were treated with indicated concentrations of pioglitazone for 16 hours. Subsequently, the concentration of PGE 2 in the cell culture medium was measured using enzyme immunoassay. In H and I, cells were treated with pioglitazone for 18 hours. Subsequently, cellular levels of cAMP (H) and PKA activity (I) were determined. A–I, mean ± SD are shown, n = 6. * , P

    Techniques Used: Expressing, Transfection, Luciferase, Activity Assay, Concentration Assay, Real-time Polymerase Chain Reaction, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay

    54) Product Images from "Identification, Purification, and Molecular Cloning of a Putative Plastidic Glucose Translocator"

    Article Title: Identification, Purification, and Molecular Cloning of a Putative Plastidic Glucose Translocator

    Journal: The Plant Cell

    doi:

    Expression Analysis of the Gene Encoding pGlcT. (A) RNA gel blot analysis of pGlcT mRNA in different tissues of tobacco. Thirty micrograms of total RNA isolated from source leaves, stems, sink leaves, roots, flower buds, and petioles was hybridized with the pGlcT cDNA probe from spinach after gel electrophoresis and subsequent transfer of the RNA to a nylon membrane. (B) Diurnal variation of the steady state concentration of pGlcT mRNA in source leaves of tobacco. Total RNA was isolated from source leaves of tobacco plants after 10 hr of light (lane 1), 1 hr of dark (lane 2), 4 hr of dark (lane 3), 12 hr of dark (lane 4), 1 hr of light (lane 5), and 3 hr of light (lane 6) during a 12-hr-light/12-hr-dark cycle. Analysis (30 μg per lane) was performed as indicated in (A) . (C) ). Analysis (50 μg per lane) was performed as in (A) .
    Figure Legend Snippet: Expression Analysis of the Gene Encoding pGlcT. (A) RNA gel blot analysis of pGlcT mRNA in different tissues of tobacco. Thirty micrograms of total RNA isolated from source leaves, stems, sink leaves, roots, flower buds, and petioles was hybridized with the pGlcT cDNA probe from spinach after gel electrophoresis and subsequent transfer of the RNA to a nylon membrane. (B) Diurnal variation of the steady state concentration of pGlcT mRNA in source leaves of tobacco. Total RNA was isolated from source leaves of tobacco plants after 10 hr of light (lane 1), 1 hr of dark (lane 2), 4 hr of dark (lane 3), 12 hr of dark (lane 4), 1 hr of light (lane 5), and 3 hr of light (lane 6) during a 12-hr-light/12-hr-dark cycle. Analysis (30 μg per lane) was performed as indicated in (A) . (C) ). Analysis (50 μg per lane) was performed as in (A) .

    Techniques Used: Expressing, Western Blot, Isolation, Nucleic Acid Electrophoresis, Concentration Assay

    55) Product Images from "Identification of an Intercistronic Internal Ribosome Entry Site in a Marek's Disease Virus Immediate-Early Gene ▿Identification of an Intercistronic Internal Ribosome Entry Site in a Marek's Disease Virus Immediate-Early Gene ▿ †"

    Article Title: Identification of an Intercistronic Internal Ribosome Entry Site in a Marek's Disease Virus Immediate-Early Gene ▿Identification of an Intercistronic Internal Ribosome Entry Site in a Marek's Disease Virus Immediate-Early Gene ▿ †

    Journal: Journal of Virology

    doi: 10.1128/JVI.02602-08

    The ICR IRES from the MDV-1 bicistronic transcript is capable of initiating translation when cap-dependent translation is inhibited. (A) List of DNA constructs used as templates to generate capped and uncapped RNA transcripts in vitro. (B) RNA transcripts
    Figure Legend Snippet: The ICR IRES from the MDV-1 bicistronic transcript is capable of initiating translation when cap-dependent translation is inhibited. (A) List of DNA constructs used as templates to generate capped and uncapped RNA transcripts in vitro. (B) RNA transcripts

    Techniques Used: Construct, In Vitro

    56) Product Images from "Direct isolation of poly(A)+ RNA from 4 M guanidine thiocyanate-lysed cell extracts using locked nucleic acid-oligo(T) capture"

    Article Title: Direct isolation of poly(A)+ RNA from 4 M guanidine thiocyanate-lysed cell extracts using locked nucleic acid-oligo(T) capture

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gnh056

    The impact of (A) GuSCN and (B) NaCl concentration on the recovery of yeast spike RNAs. ( A ) Aliquots (500 ng) of the 0.8 kb yeast THI4 spike RNA were captured at increasing final GuSCN concentrations in binding buffer from 0 to 4 M GuSCN using the reference DNA-oligo(dT) 20 and LNA_2.T affinity probes, electrophoresed in a 1% agarose gel stained with Gelstar. Lanes 1–6, THI4 spike RNA standard curve (STD) with 250, 125, 62.5, 31.3, 15.6 and 7.8 ng/lane, respectively; lanes 7–11, THI4 spike RNA captured by the reference DNA-oligo(dT) 20 in 0, 0.5, 1, 2 and 4 M GuSCN, respectively; lanes 12–16, THI4 spike RNA captured by the LNA_2.T affinity probe in 0, 0.5, 1, 2 and 4 M GuSCN, respectively. A 1 kb DNA ladder (Invitrogen, USA) was used as a size marker. Recovery (%) of the yeast THI4 RNA by the reference DNA-oligo(dT) 20 (open bars) and the LNA_2.T (solid bars) affinity probes, respectively, at increasing GuSCN concentrations was calculated by image analysis of the agarose gel. ( B ) Aliquots (1 µg) of the 0.8 kb yeast ACT1 spike RNA were captured at increasing final salt concentrations in binding buffer from 0 to 0.5 M NaCl using the reference DNA-oligo(dT) 20 and LNA_2.T affinity probes and electrophoresed in a 1% agarose gel stained with Gelstar (not shown). Recovery (%) of the yeast ACT1 RNA by the reference DNA-oligo(dT) 20 (open bars) and the LNA_2.T (solid bars) affinity probes, respectively, at different NaCl concentrations (0–0.5 M) was calculated by image analysis of the agarose gel.
    Figure Legend Snippet: The impact of (A) GuSCN and (B) NaCl concentration on the recovery of yeast spike RNAs. ( A ) Aliquots (500 ng) of the 0.8 kb yeast THI4 spike RNA were captured at increasing final GuSCN concentrations in binding buffer from 0 to 4 M GuSCN using the reference DNA-oligo(dT) 20 and LNA_2.T affinity probes, electrophoresed in a 1% agarose gel stained with Gelstar. Lanes 1–6, THI4 spike RNA standard curve (STD) with 250, 125, 62.5, 31.3, 15.6 and 7.8 ng/lane, respectively; lanes 7–11, THI4 spike RNA captured by the reference DNA-oligo(dT) 20 in 0, 0.5, 1, 2 and 4 M GuSCN, respectively; lanes 12–16, THI4 spike RNA captured by the LNA_2.T affinity probe in 0, 0.5, 1, 2 and 4 M GuSCN, respectively. A 1 kb DNA ladder (Invitrogen, USA) was used as a size marker. Recovery (%) of the yeast THI4 RNA by the reference DNA-oligo(dT) 20 (open bars) and the LNA_2.T (solid bars) affinity probes, respectively, at increasing GuSCN concentrations was calculated by image analysis of the agarose gel. ( B ) Aliquots (1 µg) of the 0.8 kb yeast ACT1 spike RNA were captured at increasing final salt concentrations in binding buffer from 0 to 0.5 M NaCl using the reference DNA-oligo(dT) 20 and LNA_2.T affinity probes and electrophoresed in a 1% agarose gel stained with Gelstar (not shown). Recovery (%) of the yeast ACT1 RNA by the reference DNA-oligo(dT) 20 (open bars) and the LNA_2.T (solid bars) affinity probes, respectively, at different NaCl concentrations (0–0.5 M) was calculated by image analysis of the agarose gel.

    Techniques Used: Concentration Assay, Binding Assay, Agarose Gel Electrophoresis, Staining, Marker

    57) Product Images from "Expression, maturation and turnover of DrrS, an unusually stable, DosR regulated small RNA in Mycobacterium tuberculosis"

    Article Title: Expression, maturation and turnover of DrrS, an unusually stable, DosR regulated small RNA in Mycobacterium tuberculosis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0174079

    5’ RACE. PCR products from wildtype and DrrS 5’ variants separated on 3% agarose. Total RNA was treated with RNA pyrophosphohydrolase (PPH) before linker ligation, cDNA synthesis and PCR amplification.
    Figure Legend Snippet: 5’ RACE. PCR products from wildtype and DrrS 5’ variants separated on 3% agarose. Total RNA was treated with RNA pyrophosphohydrolase (PPH) before linker ligation, cDNA synthesis and PCR amplification.

    Techniques Used: Polymerase Chain Reaction, Ligation, Amplification

    58) Product Images from "Receptor-Induced Dilatation in the Systemic and Intrarenal Adaptation to Pregnancy in Rats"

    Article Title: Receptor-Induced Dilatation in the Systemic and Intrarenal Adaptation to Pregnancy in Rats

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0004845

    Quantitative real-time RT-PCR for AT1, AT2, and LGR7 in the renal cortex, the aorta, and the renal artery of virgin and pregnant rats. Individual samples were normalized to β-actin mRNA levels and data are expressed as the mRNA expression relative to the virgin group. Values represent the mean±SEM of six samples/group. *p
    Figure Legend Snippet: Quantitative real-time RT-PCR for AT1, AT2, and LGR7 in the renal cortex, the aorta, and the renal artery of virgin and pregnant rats. Individual samples were normalized to β-actin mRNA levels and data are expressed as the mRNA expression relative to the virgin group. Values represent the mean±SEM of six samples/group. *p

    Techniques Used: Quantitative RT-PCR, Expressing

    Quantitative real-time RT-PCR for the Ang II receptors (AT1 and AT2), the relaxin receptor (LGR7), and iNOS in MCs isolated from virgin and pregnant rats. Total RNA was isolated from pooled cells that were obtained from three or four culture flasks from each group. Individual samples were normalized to β-actin mRNA levels and the data are expressed as the mRNA expression relative to the virgin group. Values represent the mean±SEM of six samples/group. *p
    Figure Legend Snippet: Quantitative real-time RT-PCR for the Ang II receptors (AT1 and AT2), the relaxin receptor (LGR7), and iNOS in MCs isolated from virgin and pregnant rats. Total RNA was isolated from pooled cells that were obtained from three or four culture flasks from each group. Individual samples were normalized to β-actin mRNA levels and the data are expressed as the mRNA expression relative to the virgin group. Values represent the mean±SEM of six samples/group. *p

    Techniques Used: Quantitative RT-PCR, Isolation, Expressing

    59) Product Images from "Investigating the specificity and stoichiometry of RNA binding by the nucleocapsid protein of Bunyamwera virus"

    Article Title: Investigating the specificity and stoichiometry of RNA binding by the nucleocapsid protein of Bunyamwera virus

    Journal: RNA

    doi: 10.1261/rna.1367209

    Schematics of the most energetically favorable secondary structures predicted by M-fold to form in the 5′ end of the ( A ) genomic and ( B ) anti-genomic strands of the BUNV S segment. The terminal strand of each nucleotide is circled.
    Figure Legend Snippet: Schematics of the most energetically favorable secondary structures predicted by M-fold to form in the 5′ end of the ( A ) genomic and ( B ) anti-genomic strands of the BUNV S segment. The terminal strand of each nucleotide is circled.

    Techniques Used:

    60) Product Images from "Telmisartan attenuates hepatic fibrosis in bile duct-ligated rats"

    Article Title: Telmisartan attenuates hepatic fibrosis in bile duct-ligated rats

    Journal: Acta Pharmacologica Sinica

    doi: 10.1038/aps.2012.115

    (A) Relative expression of hepatic ACE2, MAS, ACE, AT1-R, col III, and TGF-β1 by real-time PCR. Expression levels were normalized to GAPDH. (B) Representative DNA gel electrophoresis for quantitation of mRNA expression. Lane 1, 4, 7, 10, 13, 16,
    Figure Legend Snippet: (A) Relative expression of hepatic ACE2, MAS, ACE, AT1-R, col III, and TGF-β1 by real-time PCR. Expression levels were normalized to GAPDH. (B) Representative DNA gel electrophoresis for quantitation of mRNA expression. Lane 1, 4, 7, 10, 13, 16,

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, DNA Gel Electrophoresis, Quantitation Assay

    Western blot analysis of ACE2, MAS, ACE, AT1-R, col III, and TGF-β1
    Figure Legend Snippet: Western blot analysis of ACE2, MAS, ACE, AT1-R, col III, and TGF-β1

    Techniques Used: Western Blot

    61) Product Images from "The mRNA cap methyltransferase gene TbCMT1 is not essential in vitro but is a virulence factor in vivo for bloodstream form Trypanosoma brucei"

    Article Title: The mRNA cap methyltransferase gene TbCMT1 is not essential in vitro but is a virulence factor in vivo for bloodstream form Trypanosoma brucei

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0201263

    TbCMT1 is not required for T . brucei Lister 427 BSF proliferation in cell culture. (A) Total RNA was purified from TbCMT1 conditional null cells cultured with (+tet) and without tetracycline for 24, 48 and 72 h. TbCMT1 expression was analysed by qRT-PCR normalised to telomerase reverse transcriptase ( TERT ). The delta Ct values (dCt) for nine measurements for each condition are visualised as a swarm plot to show all observations along with representations of the underlying distributions. (B) Cumulative cell counts of triplicate TbCMT1 conditional null mutant cell cultures grown with (plus) and without (minus) tetracycline. (C) Cumulative cell counts of triplicate wild type (WT) and TbCMT1 null cells cultured in parallel. For the data in panels B and C, the cell counts of three biological replicates are reported after 2, 4, 6, 8 and 10 days. The cultures were counted and diluted to 10 5 cells/ml every two days in (B) and to 10 4 cells/ml every two days in (C). The cell counts are reported as the log10 value of the cumulative number of parasites per ml of cell culture allowing for the aforementioned dilution factors.
    Figure Legend Snippet: TbCMT1 is not required for T . brucei Lister 427 BSF proliferation in cell culture. (A) Total RNA was purified from TbCMT1 conditional null cells cultured with (+tet) and without tetracycline for 24, 48 and 72 h. TbCMT1 expression was analysed by qRT-PCR normalised to telomerase reverse transcriptase ( TERT ). The delta Ct values (dCt) for nine measurements for each condition are visualised as a swarm plot to show all observations along with representations of the underlying distributions. (B) Cumulative cell counts of triplicate TbCMT1 conditional null mutant cell cultures grown with (plus) and without (minus) tetracycline. (C) Cumulative cell counts of triplicate wild type (WT) and TbCMT1 null cells cultured in parallel. For the data in panels B and C, the cell counts of three biological replicates are reported after 2, 4, 6, 8 and 10 days. The cultures were counted and diluted to 10 5 cells/ml every two days in (B) and to 10 4 cells/ml every two days in (C). The cell counts are reported as the log10 value of the cumulative number of parasites per ml of cell culture allowing for the aforementioned dilution factors.

    Techniques Used: Cell Culture, Purification, Expressing, Quantitative RT-PCR, Mutagenesis

    62) Product Images from "Transcriptome-wide mapping of m6A and m6Am at single-nucleotide resolution using miCLIP"

    Article Title: Transcriptome-wide mapping of m6A and m6Am at single-nucleotide resolution using miCLIP

    Journal: Current protocols in molecular biology

    doi: 10.1002/cpmb.88

    Schematic of the miCLIP protocol. Capped (grey circle) and polyadenylated cellular RNA containing both m 6 Am (red triangle) and m 6 A (red circle) is fragmented and incubated with an anti-m 6 A antibody. Following UV-crosslinking, the antibody-RNA complexes are recovered using protein A/G-affinity beads and a 3’-adapter is ligated to the RNA. Antibody-RNA complexes are then purified by transferring to a nitrocellulose membrane and eluted using proteinase K, leaving only a small peptide fragment crosslinked at the m 6 A/m 6 Am site. RNA fragments are reverse transcribed, which results in mutations or truncations at the crosslink site in the resulting cDNA. Finally, the cDNA is circularized, re-linearized and amplified by PCR to generate final libraries for sequencing.
    Figure Legend Snippet: Schematic of the miCLIP protocol. Capped (grey circle) and polyadenylated cellular RNA containing both m 6 Am (red triangle) and m 6 A (red circle) is fragmented and incubated with an anti-m 6 A antibody. Following UV-crosslinking, the antibody-RNA complexes are recovered using protein A/G-affinity beads and a 3’-adapter is ligated to the RNA. Antibody-RNA complexes are then purified by transferring to a nitrocellulose membrane and eluted using proteinase K, leaving only a small peptide fragment crosslinked at the m 6 A/m 6 Am site. RNA fragments are reverse transcribed, which results in mutations or truncations at the crosslink site in the resulting cDNA. Finally, the cDNA is circularized, re-linearized and amplified by PCR to generate final libraries for sequencing.

    Techniques Used: Incubation, Purification, Transferring, Amplification, Polymerase Chain Reaction, Sequencing

    63) Product Images from "Regulation of the human tyrosinase gene in retinal pigment epithelium cells: the significance of transcription factor orthodenticle homeobox 2 and its polymorphic binding site"

    Article Title: Regulation of the human tyrosinase gene in retinal pigment epithelium cells: the significance of transcription factor orthodenticle homeobox 2 and its polymorphic binding site

    Journal: Molecular Vision

    doi:

    Expression of TYR , MITF-A , MITF-D , MITF-H , MITF-M , and OTX2 mRNA in RPE cells. ARPE-19 mRNA was collected at various time points (6 h, 24 h, 48 h, 96 h, 14 days, and 30 days). Other samples were isolated at 96 h from D407 RPE cells and G-361 melanoma cells, a positive control for TYR expression. Results are presented as the mean normalized expression±SD relative to 6 h ARPE-19 culture (=1) from three to four independent cultures each performed in triplicate. Due to very low expression levels, MITF-D values were normalized relative to MITF-H (6 h ARPE-19 culture=1).
    Figure Legend Snippet: Expression of TYR , MITF-A , MITF-D , MITF-H , MITF-M , and OTX2 mRNA in RPE cells. ARPE-19 mRNA was collected at various time points (6 h, 24 h, 48 h, 96 h, 14 days, and 30 days). Other samples were isolated at 96 h from D407 RPE cells and G-361 melanoma cells, a positive control for TYR expression. Results are presented as the mean normalized expression±SD relative to 6 h ARPE-19 culture (=1) from three to four independent cultures each performed in triplicate. Due to very low expression levels, MITF-D values were normalized relative to MITF-H (6 h ARPE-19 culture=1).

    Techniques Used: Expressing, Isolation, Positive Control

    64) Product Images from "A transcriptome-wide, organ-specific regulatory map of Dendrobium officinale, an important traditional Chinese orchid herb"

    Article Title: A transcriptome-wide, organ-specific regulatory map of Dendrobium officinale, an important traditional Chinese orchid herb

    Journal: Scientific Reports

    doi: 10.1038/srep18864

    Tandemly distributed small RNAs (sRNAs) identified on the highly structured microRNA (miRNA) precursor candidates in Dendrobium officinale . ( A ) The transcript comp124801_c0_seq1 assembled by RNA-seq reads could form an internal hairpin structure with a long-stem region (partially delineated by a pink box). In addition to generating miRNAs (dof-miR340, dof-miR341, dof-miR1002 and dof-miR1004) and miRNA*s (dof-miR1002* and dof-miR1004*), the long-stem region potentially encodes three pairs of tandemly distributed sRNAs (124801_sRNA1 and 124801_sRNA6, 124801_sRNA2 and 124801_sRNA5, and 124801_sRNA3 and 124801_sRNA4). Each pair possesses 2-nt 3’ overhangs. Five degradome signatures (124801_degr1 to 124801_degr5) were detected at the ends of certain tandemly distributed sRNAs. And, 124801_degr3 also appeared at the 5’ ends of dof-miR-340 and dof-miR-341, and 124801_degr4 and 124801_degr5 are present at the 3’ ends of dof-miR-1004. The accumulation levels (normalized in RPM, reads per million; please refer to Materials and Methods for RPM calculation) of the degradome signatures, the miRNAs, the miRNA*s and the tandemly distributed sRNAs are shown in the diagrams on the right of the panel. Their accumulation levels in the stems of Dendrobium officinale were highlighted in pink background color. ( B ) The transcript comp168357_c1_seq6 assembled by RNA-seq reads could form an internal hairpin structure with a long-stem region (partially included in a pink box). Within this region, three pairs of sRNAs (including 168357_sRNA2 and 168357_sRNA6, 168357_sRNA3 and 168357_sRNA5, and the dof-miR-1023/dof-miR-1023* duplex) along with two unpaired sRNAs (168357_sRNA1 and 168357_sRNA4) were identified to be distributed tandemly. Each pair possesses 2-nt 3’ overhangs. Eleven degradome signatures (168357_degr1 to 168357_degr11) were detected at the ends of certain tandemly distributed sRNAs. The accumulation levels (in RPM) of the degradome signatures, the miRNAs, the miRNA*s and the tandemly distributed sRNAs are shown in the diagrams on the right of the panel. The secondary structures of the two transcripts were predicted by using RNAfold ( http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi ) 22 .
    Figure Legend Snippet: Tandemly distributed small RNAs (sRNAs) identified on the highly structured microRNA (miRNA) precursor candidates in Dendrobium officinale . ( A ) The transcript comp124801_c0_seq1 assembled by RNA-seq reads could form an internal hairpin structure with a long-stem region (partially delineated by a pink box). In addition to generating miRNAs (dof-miR340, dof-miR341, dof-miR1002 and dof-miR1004) and miRNA*s (dof-miR1002* and dof-miR1004*), the long-stem region potentially encodes three pairs of tandemly distributed sRNAs (124801_sRNA1 and 124801_sRNA6, 124801_sRNA2 and 124801_sRNA5, and 124801_sRNA3 and 124801_sRNA4). Each pair possesses 2-nt 3’ overhangs. Five degradome signatures (124801_degr1 to 124801_degr5) were detected at the ends of certain tandemly distributed sRNAs. And, 124801_degr3 also appeared at the 5’ ends of dof-miR-340 and dof-miR-341, and 124801_degr4 and 124801_degr5 are present at the 3’ ends of dof-miR-1004. The accumulation levels (normalized in RPM, reads per million; please refer to Materials and Methods for RPM calculation) of the degradome signatures, the miRNAs, the miRNA*s and the tandemly distributed sRNAs are shown in the diagrams on the right of the panel. Their accumulation levels in the stems of Dendrobium officinale were highlighted in pink background color. ( B ) The transcript comp168357_c1_seq6 assembled by RNA-seq reads could form an internal hairpin structure with a long-stem region (partially included in a pink box). Within this region, three pairs of sRNAs (including 168357_sRNA2 and 168357_sRNA6, 168357_sRNA3 and 168357_sRNA5, and the dof-miR-1023/dof-miR-1023* duplex) along with two unpaired sRNAs (168357_sRNA1 and 168357_sRNA4) were identified to be distributed tandemly. Each pair possesses 2-nt 3’ overhangs. Eleven degradome signatures (168357_degr1 to 168357_degr11) were detected at the ends of certain tandemly distributed sRNAs. The accumulation levels (in RPM) of the degradome signatures, the miRNAs, the miRNA*s and the tandemly distributed sRNAs are shown in the diagrams on the right of the panel. The secondary structures of the two transcripts were predicted by using RNAfold ( http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi ) 22 .

    Techniques Used: RNA Sequencing Assay

    65) Product Images from "RNA Silencing of Single and Multiple Members in a Gene Family of Rice 1RNA Silencing of Single and Multiple Members in a Gene Family of Rice 1 [w]"

    Article Title: RNA Silencing of Single and Multiple Members in a Gene Family of Rice 1RNA Silencing of Single and Multiple Members in a Gene Family of Rice 1 [w]

    Journal: Plant Physiology

    doi: 10.1104/pp.105.063933

    RNA silencing of the endogenous PDS gene. A, Schematic representation of the PDS gene and IR construct. The central 470 bp (shaded region, nt 1,260–1,730; also used as trigger probe) of the 2,027-bp PDS cDNA was used as an RNA silencing trigger.
    Figure Legend Snippet: RNA silencing of the endogenous PDS gene. A, Schematic representation of the PDS gene and IR construct. The central 470 bp (shaded region, nt 1,260–1,730; also used as trigger probe) of the 2,027-bp PDS cDNA was used as an RNA silencing trigger.

    Techniques Used: Construct

    66) Product Images from "Expression, maturation and turnover of DrrS, an unusually stable, DosR regulated small RNA in Mycobacterium tuberculosis"

    Article Title: Expression, maturation and turnover of DrrS, an unusually stable, DosR regulated small RNA in Mycobacterium tuberculosis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0174079

    5’ RACE. PCR products from wildtype and DrrS 5’ variants separated on 3% agarose. Total RNA was treated with RNA pyrophosphohydrolase (PPH) before linker ligation, cDNA synthesis and PCR amplification.
    Figure Legend Snippet: 5’ RACE. PCR products from wildtype and DrrS 5’ variants separated on 3% agarose. Total RNA was treated with RNA pyrophosphohydrolase (PPH) before linker ligation, cDNA synthesis and PCR amplification.

    Techniques Used: Polymerase Chain Reaction, Ligation, Amplification

    67) Product Images from "Expression, maturation and turnover of DrrS, an unusually stable, DosR regulated small RNA in Mycobacterium tuberculosis"

    Article Title: Expression, maturation and turnover of DrrS, an unusually stable, DosR regulated small RNA in Mycobacterium tuberculosis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0174079

    5’ RACE. PCR products from wildtype and DrrS 5’ variants separated on 3% agarose. Total RNA was treated with RNA pyrophosphohydrolase (PPH) before linker ligation, cDNA synthesis and PCR amplification.
    Figure Legend Snippet: 5’ RACE. PCR products from wildtype and DrrS 5’ variants separated on 3% agarose. Total RNA was treated with RNA pyrophosphohydrolase (PPH) before linker ligation, cDNA synthesis and PCR amplification.

    Techniques Used: Polymerase Chain Reaction, Ligation, Amplification

    68) Product Images from "Differential Expression Analysis of Olfactory Genes Based on a Combination of Sequencing Platforms and Behavioral Investigations in Aphidius gifuensis"

    Article Title: Differential Expression Analysis of Olfactory Genes Based on a Combination of Sequencing Platforms and Behavioral Investigations in Aphidius gifuensis

    Journal: Frontiers in Physiology

    doi: 10.3389/fphys.2018.01679

    Venn diagram based on a combined analysis of differential expression of transcripts and behavioral investigations. (A,B) According to the behavioral test results for the response to wasps of the opposite sex, the intersection of differentially expressed olfactory genes in VM/VF and comparable olfactory genes in MM/VF contains five genes (upregulated) and one gene (downregulated), which represent six candidate genes that may be involved in the recognition of the opposite sex by VMs. The intersection of differentially expressed olfactory genes in MF/VF and comparable olfactory genes in MM/VF contains three genes (upregulated) and four genes (downregulated), respectively, which represents seven candidate genes that could be involved in the recognition of the opposite sex by MFs. (C,D) According to the results of the behavioral test for the response to EBF, the number of common up- and downregulated olfactory genes was 1 and 3, respectively, which represent four candidate genes that may be involved in EBF perception.
    Figure Legend Snippet: Venn diagram based on a combined analysis of differential expression of transcripts and behavioral investigations. (A,B) According to the behavioral test results for the response to wasps of the opposite sex, the intersection of differentially expressed olfactory genes in VM/VF and comparable olfactory genes in MM/VF contains five genes (upregulated) and one gene (downregulated), which represent six candidate genes that may be involved in the recognition of the opposite sex by VMs. The intersection of differentially expressed olfactory genes in MF/VF and comparable olfactory genes in MM/VF contains three genes (upregulated) and four genes (downregulated), respectively, which represents seven candidate genes that could be involved in the recognition of the opposite sex by MFs. (C,D) According to the results of the behavioral test for the response to EBF, the number of common up- and downregulated olfactory genes was 1 and 3, respectively, which represent four candidate genes that may be involved in EBF perception.

    Techniques Used: Expressing

    Heatmap of all the annotated olfactory genes. Genes marked in green, three of six candidate genes involved in recognition of the opposite sex by VMs; blue, six candidate genes involved in recognition of the opposite sex by MFs; red, the remaining four of seven candidate genes involved in recognition of the opposite sex by VMs, which are also involved in EBF perception.
    Figure Legend Snippet: Heatmap of all the annotated olfactory genes. Genes marked in green, three of six candidate genes involved in recognition of the opposite sex by VMs; blue, six candidate genes involved in recognition of the opposite sex by MFs; red, the remaining four of seven candidate genes involved in recognition of the opposite sex by VMs, which are also involved in EBF perception.

    Techniques Used:

    69) Product Images from "Direct isolation of poly(A)+ RNA from 4 M guanidine thiocyanate-lysed cell extracts using locked nucleic acid-oligo(T) capture"

    Article Title: Direct isolation of poly(A)+ RNA from 4 M guanidine thiocyanate-lysed cell extracts using locked nucleic acid-oligo(T) capture

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gnh056

    The impact of (A) GuSCN and (B) NaCl concentration on the recovery of yeast spike RNAs. ( A ) Aliquots (500 ng) of the 0.8 kb yeast THI4 spike RNA were captured at increasing final GuSCN concentrations in binding buffer from 0 to 4 M GuSCN using the reference DNA-oligo(dT) 20 and LNA_2.T affinity probes, electrophoresed in a 1% agarose gel stained with Gelstar. Lanes 1–6, THI4 spike RNA standard curve (STD) with 250, 125, 62.5, 31.3, 15.6 and 7.8 ng/lane, respectively; lanes 7–11, THI4 spike RNA captured by the reference DNA-oligo(dT) 20 in 0, 0.5, 1, 2 and 4 M GuSCN, respectively; lanes 12–16, THI4 spike RNA captured by the LNA_2.T affinity probe in 0, 0.5, 1, 2 and 4 M GuSCN, respectively. A 1 kb DNA ladder (Invitrogen, USA) was used as a size marker. Recovery (%) of the yeast THI4 RNA by the reference DNA-oligo(dT) 20 (open bars) and the LNA_2.T (solid bars) affinity probes, respectively, at increasing GuSCN concentrations was calculated by image analysis of the agarose gel. ( B ) Aliquots (1 µg) of the 0.8 kb yeast ACT1 spike RNA were captured at increasing final salt concentrations in binding buffer from 0 to 0.5 M NaCl using the reference DNA-oligo(dT) 20 and LNA_2.T affinity probes and electrophoresed in a 1% agarose gel stained with Gelstar (not shown). Recovery (%) of the yeast ACT1 RNA by the reference DNA-oligo(dT) 20 (open bars) and the LNA_2.T (solid bars) affinity probes, respectively, at different NaCl concentrations (0–0.5 M) was calculated by image analysis of the agarose gel.
    Figure Legend Snippet: The impact of (A) GuSCN and (B) NaCl concentration on the recovery of yeast spike RNAs. ( A ) Aliquots (500 ng) of the 0.8 kb yeast THI4 spike RNA were captured at increasing final GuSCN concentrations in binding buffer from 0 to 4 M GuSCN using the reference DNA-oligo(dT) 20 and LNA_2.T affinity probes, electrophoresed in a 1% agarose gel stained with Gelstar. Lanes 1–6, THI4 spike RNA standard curve (STD) with 250, 125, 62.5, 31.3, 15.6 and 7.8 ng/lane, respectively; lanes 7–11, THI4 spike RNA captured by the reference DNA-oligo(dT) 20 in 0, 0.5, 1, 2 and 4 M GuSCN, respectively; lanes 12–16, THI4 spike RNA captured by the LNA_2.T affinity probe in 0, 0.5, 1, 2 and 4 M GuSCN, respectively. A 1 kb DNA ladder (Invitrogen, USA) was used as a size marker. Recovery (%) of the yeast THI4 RNA by the reference DNA-oligo(dT) 20 (open bars) and the LNA_2.T (solid bars) affinity probes, respectively, at increasing GuSCN concentrations was calculated by image analysis of the agarose gel. ( B ) Aliquots (1 µg) of the 0.8 kb yeast ACT1 spike RNA were captured at increasing final salt concentrations in binding buffer from 0 to 0.5 M NaCl using the reference DNA-oligo(dT) 20 and LNA_2.T affinity probes and electrophoresed in a 1% agarose gel stained with Gelstar (not shown). Recovery (%) of the yeast ACT1 RNA by the reference DNA-oligo(dT) 20 (open bars) and the LNA_2.T (solid bars) affinity probes, respectively, at different NaCl concentrations (0–0.5 M) was calculated by image analysis of the agarose gel.

    Techniques Used: Concentration Assay, Binding Assay, Agarose Gel Electrophoresis, Staining, Marker

    Northern blot analysis of yeast poly(A) + RNA isolated by the LNA_2.T affinity probe under low salt binding conditions. Yeast poly(A) + RNA was isolated from wild type, wild type heat shocked and Δ hsp78 mutant heat shocked total RNA and probed sequentially with 32 P-labelled fragments for the yeast HSP78 and ACT1 genes, respectively.
    Figure Legend Snippet: Northern blot analysis of yeast poly(A) + RNA isolated by the LNA_2.T affinity probe under low salt binding conditions. Yeast poly(A) + RNA was isolated from wild type, wild type heat shocked and Δ hsp78 mutant heat shocked total RNA and probed sequentially with 32 P-labelled fragments for the yeast HSP78 and ACT1 genes, respectively.

    Techniques Used: Northern Blot, Isolation, Binding Assay, Mutagenesis

    70) Product Images from "T Lymphocytes Promote the Antiviral and Inflammatory Responses of Airway Epithelial Cells"

    Article Title: T Lymphocytes Promote the Antiviral and Inflammatory Responses of Airway Epithelial Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0026293

    Epithelial CXCL10 mRNA induction is proportional to the amounts of IFNγ and TNF produced by T cell clones. HNEC were co-cultured with T cell clones obtained from PBMC of a normal individual. CXCL10 mRNA induction is expressed as a function of IFNγ (A) and TNF (B) secreted by T cell clones in the culture medium. Epithelial CXCL10 mRNAs are measured by real-time PCR and expressed as fold increase relative to those measured in epithelial cells cultured without T cells. IFNγ and TNF were measured by TenPlex bead immunoassay and ELISA.
    Figure Legend Snippet: Epithelial CXCL10 mRNA induction is proportional to the amounts of IFNγ and TNF produced by T cell clones. HNEC were co-cultured with T cell clones obtained from PBMC of a normal individual. CXCL10 mRNA induction is expressed as a function of IFNγ (A) and TNF (B) secreted by T cell clones in the culture medium. Epithelial CXCL10 mRNAs are measured by real-time PCR and expressed as fold increase relative to those measured in epithelial cells cultured without T cells. IFNγ and TNF were measured by TenPlex bead immunoassay and ELISA.

    Techniques Used: Produced, Clone Assay, Cell Culture, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Epithelial CXCL10 mRNA is induced by HRV14 and T cells, CXCL8 and IL-6 mRNA are induced only by T cells. HNEC were exposed apically to HRV14 alone (HRV), or co-cultured with activated T cells alone (T cells), or exposed to HRV14 washed thoroughly then co-cultured with activated T cells (HRV+T cells). The expression of CXCL10 mRNA (A), CXCL8 mRNA (B) and IL-6 mRNA (C) was determined by real-time PCR. Data represent the fold increase relative to non-infected cells cultured in the absence of T cells. Shown are the boxplots with the 25 th , 50 th (median), 75 th percentiles, the mean (open square), the maximum and the minimum values. *p
    Figure Legend Snippet: Epithelial CXCL10 mRNA is induced by HRV14 and T cells, CXCL8 and IL-6 mRNA are induced only by T cells. HNEC were exposed apically to HRV14 alone (HRV), or co-cultured with activated T cells alone (T cells), or exposed to HRV14 washed thoroughly then co-cultured with activated T cells (HRV+T cells). The expression of CXCL10 mRNA (A), CXCL8 mRNA (B) and IL-6 mRNA (C) was determined by real-time PCR. Data represent the fold increase relative to non-infected cells cultured in the absence of T cells. Shown are the boxplots with the 25 th , 50 th (median), 75 th percentiles, the mean (open square), the maximum and the minimum values. *p

    Techniques Used: Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Infection

    Anti-IFNγ and TNF soluble receptor p75 strongly reduce epithelial CXCL10 mRNA expression but only partially attenuate epithelial NOS2 expression induced by T cells. HNEC were co-cultured with activated T cells in the absence or presence of anti-IFNγ, TNF soluble receptor (srTNF) or an isotype-matched antibody (IgG). CXCL10 (A) and NOS2 (B) mRNA were measured by real-time PCR. Data are presented as fold change relative to those obtained in the absence of neutralizing agents. Shown are the boxplots with the 25 th , 50 th (median), 75 th percentiles, the mean (open square), the maximum and the minimum values. *p
    Figure Legend Snippet: Anti-IFNγ and TNF soluble receptor p75 strongly reduce epithelial CXCL10 mRNA expression but only partially attenuate epithelial NOS2 expression induced by T cells. HNEC were co-cultured with activated T cells in the absence or presence of anti-IFNγ, TNF soluble receptor (srTNF) or an isotype-matched antibody (IgG). CXCL10 (A) and NOS2 (B) mRNA were measured by real-time PCR. Data are presented as fold change relative to those obtained in the absence of neutralizing agents. Shown are the boxplots with the 25 th , 50 th (median), 75 th percentiles, the mean (open square), the maximum and the minimum values. *p

    Techniques Used: Expressing, Cell Culture, Real-time Polymerase Chain Reaction

    71) Product Images from "Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing"

    Article Title: Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr693

    The two terminal 3′ bases of the 5′ adapter are the primary determinants of T4-RNA ligase 1 (Rnl1) ligation efficiency. Two distinct sets of 5′ adapters, one consisting of adapters with mixed bases in the last four bases (fNNNN) and another consisting of adapters with mixed bases in the last two bases (fNN), were used to generate a miRNA derived cDNA library for ( A ) human 293T and ( B ) mouse embryonic stem cell lines. miRNA abundance in read counts (dots) were plotted; the fNNNN data were compressed to NN, by combining values for AANN through TTNN for each NN. The high correlation between the compressed fNNNN and the fNN datasets indicates that the two terminal bases are dominant determinants of ligation efficiency. There are exceptions shown in red, which are systematic differences (106b, 181 in 293T cell), which we detected in an independent experiment described in Figure 2 suggesting that this is not a stochastic effect. The naming convention in all our figures is to show the beginning and end of the sequence followed by an m (for a canonical mature) or n for a non-canonical miRNA sequence followed by the name of the miRNA. Thus in the left we have a canonical mature hsa-miR-106-b and a non-canonical hsa-miR-218. The high abundance for hsa-miR-106b suggested by the fNNNN strategy (in contrast to the low values suggested by fNN and other strategies) seems real, as the microarray and RT–PCR results ( Figure 9 ) are in concordance with the fNNNN values.
    Figure Legend Snippet: The two terminal 3′ bases of the 5′ adapter are the primary determinants of T4-RNA ligase 1 (Rnl1) ligation efficiency. Two distinct sets of 5′ adapters, one consisting of adapters with mixed bases in the last four bases (fNNNN) and another consisting of adapters with mixed bases in the last two bases (fNN), were used to generate a miRNA derived cDNA library for ( A ) human 293T and ( B ) mouse embryonic stem cell lines. miRNA abundance in read counts (dots) were plotted; the fNNNN data were compressed to NN, by combining values for AANN through TTNN for each NN. The high correlation between the compressed fNNNN and the fNN datasets indicates that the two terminal bases are dominant determinants of ligation efficiency. There are exceptions shown in red, which are systematic differences (106b, 181 in 293T cell), which we detected in an independent experiment described in Figure 2 suggesting that this is not a stochastic effect. The naming convention in all our figures is to show the beginning and end of the sequence followed by an m (for a canonical mature) or n for a non-canonical miRNA sequence followed by the name of the miRNA. Thus in the left we have a canonical mature hsa-miR-106-b and a non-canonical hsa-miR-218. The high abundance for hsa-miR-106b suggested by the fNNNN strategy (in contrast to the low values suggested by fNN and other strategies) seems real, as the microarray and RT–PCR results ( Figure 9 ) are in concordance with the fNNNN values.

    Techniques Used: Ligation, Derivative Assay, cDNA Library Assay, Sequencing, Microarray, Reverse Transcription Polymerase Chain Reaction

    Comparison of sequencing against microarray ( A and B ) and RT–PCR ( C and D ) for mES (B and D) and 293T (A and C). There are outliers, such as miR-106b, which are only captured by the fNNNN strategy, but overall, there is significant correlation between the fNN_eNN strategy and the microarray data (A) and the fNN_eNN strategy and the RT–PCR data (C), while the fNN sequencing strategy does not give a good correlation to RT–PCR and array data (B and D).
    Figure Legend Snippet: Comparison of sequencing against microarray ( A and B ) and RT–PCR ( C and D ) for mES (B and D) and 293T (A and C). There are outliers, such as miR-106b, which are only captured by the fNNNN strategy, but overall, there is significant correlation between the fNN_eNN strategy and the microarray data (A) and the fNN_eNN strategy and the RT–PCR data (C), while the fNN sequencing strategy does not give a good correlation to RT–PCR and array data (B and D).

    Techniques Used: Sequencing, Microarray, Reverse Transcription Polymerase Chain Reaction

    72) Product Images from "Unproductive alternative splicing and nonsense mRNAs: A widespread phenomenon among plant circadian clock genes"

    Article Title: Unproductive alternative splicing and nonsense mRNAs: A widespread phenomenon among plant circadian clock genes

    Journal: Biology Direct

    doi: 10.1186/1745-6150-7-20

    Regulation of the expression levels of alternatively spliced CCA1 isoforms by environmental stress. ( A ) Semi-quantitative RT-PCR analysis of the I4R event in CCA1 transcripts under different abiotic stress treatments. The relative abundance of the PTC-harboring CCA1 I4R isoform changes compared to the full-length spliced variant (I4S) after cold stress treatment. Two-week-old seedlings were treated as previously described [ 2 ]. Ctrl - untreated seedlings, Hi light - high intensity light, Heat - heat stress (42 °C), Cold - cold stress (4 °C), Salt - high salinity (0.5 M sodium chloride), Desiccation - dehydration (polyethylene glycol treatment). eF1α mRNA was used to demonstrate an equal PCR amplification of cDNAs. PCR was carried out for 16 cycles for all the reactions. PCR products were separated in 2 % agarose gels and stained by ethidium bromide. ( B ) qRT-PCR analysis of cold stress-induced changes in relative abundance of the CCA1 splice variants. The normalized expression of the CCA1 transcripts with spliced intron 4 (CCA1 I4S) increased after 12 and 168 hours of the cold treatment (4 °C). In contrast, the normalized fold expression of the I4R transcripts sharply decreased following the treatment at 4 °C. GOG was used as a reference housekeeping transcript. The sampling and conditions of the time course are described in detail in the Methods. The normalized fold change of expression of the target transcripts was calculated using a –ΔΔCt method and CFX Manager software. Vertical bars denote the standard error of the mean. Both RT-PCR and qRT-PCR were performed using splicing event-specific oligonucleotide primers (see Additional file 2 ). ZT - Zeitgeber time (hours).
    Figure Legend Snippet: Regulation of the expression levels of alternatively spliced CCA1 isoforms by environmental stress. ( A ) Semi-quantitative RT-PCR analysis of the I4R event in CCA1 transcripts under different abiotic stress treatments. The relative abundance of the PTC-harboring CCA1 I4R isoform changes compared to the full-length spliced variant (I4S) after cold stress treatment. Two-week-old seedlings were treated as previously described [ 2 ]. Ctrl - untreated seedlings, Hi light - high intensity light, Heat - heat stress (42 °C), Cold - cold stress (4 °C), Salt - high salinity (0.5 M sodium chloride), Desiccation - dehydration (polyethylene glycol treatment). eF1α mRNA was used to demonstrate an equal PCR amplification of cDNAs. PCR was carried out for 16 cycles for all the reactions. PCR products were separated in 2 % agarose gels and stained by ethidium bromide. ( B ) qRT-PCR analysis of cold stress-induced changes in relative abundance of the CCA1 splice variants. The normalized expression of the CCA1 transcripts with spliced intron 4 (CCA1 I4S) increased after 12 and 168 hours of the cold treatment (4 °C). In contrast, the normalized fold expression of the I4R transcripts sharply decreased following the treatment at 4 °C. GOG was used as a reference housekeeping transcript. The sampling and conditions of the time course are described in detail in the Methods. The normalized fold change of expression of the target transcripts was calculated using a –ΔΔCt method and CFX Manager software. Vertical bars denote the standard error of the mean. Both RT-PCR and qRT-PCR were performed using splicing event-specific oligonucleotide primers (see Additional file 2 ). ZT - Zeitgeber time (hours).

    Techniques Used: Expressing, Quantitative RT-PCR, Variant Assay, Polymerase Chain Reaction, Amplification, Staining, Sampling, Software, Reverse Transcription Polymerase Chain Reaction

    Alternative splicing of RVE2 pre-mRNA introduces an in-frame nonsense codon via a PCE event. ( A ) The schematic representation of the AS event in intron 1 introducing a PCE in the RVE2 transcript. The 5' UTR is shown by a light box, protein coding exons and the PCE are indicated by dark and hatched boxes, respectively. Binding sites for primers F1-R1 and F2-R2 used in RT-PCR are shown by arrows. Normal and premature termination codons are shown by the top and bottom stars. Dashed lines indicate primer portions spanning splice junctions. The gene model is not drawn to scale. ( B ) The alignment of the 5’ portion of RVE2 cDNA (top) and Sanger sequences of the PCR products (bottom). The PCE sequence is highlighted in grey. The initiation codon is boxed. ( C ) Quantification of alternatively spliced RVE2 mRNA harboring PCE event using qRT-PCR. The diurnal conditions and the sampling scheme are described in detail in Methods. Arrows indicate the sampling time points (ZT, hours). The relative transcript quantities were calculated using the –ΔΔCt method and CFX Manager software (BioRad). GOG mRNA was used as an internal reference. Vertical bars represent the standard error of the mean.
    Figure Legend Snippet: Alternative splicing of RVE2 pre-mRNA introduces an in-frame nonsense codon via a PCE event. ( A ) The schematic representation of the AS event in intron 1 introducing a PCE in the RVE2 transcript. The 5' UTR is shown by a light box, protein coding exons and the PCE are indicated by dark and hatched boxes, respectively. Binding sites for primers F1-R1 and F2-R2 used in RT-PCR are shown by arrows. Normal and premature termination codons are shown by the top and bottom stars. Dashed lines indicate primer portions spanning splice junctions. The gene model is not drawn to scale. ( B ) The alignment of the 5’ portion of RVE2 cDNA (top) and Sanger sequences of the PCR products (bottom). The PCE sequence is highlighted in grey. The initiation codon is boxed. ( C ) Quantification of alternatively spliced RVE2 mRNA harboring PCE event using qRT-PCR. The diurnal conditions and the sampling scheme are described in detail in Methods. Arrows indicate the sampling time points (ZT, hours). The relative transcript quantities were calculated using the –ΔΔCt method and CFX Manager software (BioRad). GOG mRNA was used as an internal reference. Vertical bars represent the standard error of the mean.

    Techniques Used: Binding Assay, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Sequencing, Quantitative RT-PCR, Sampling, Software

    73) Product Images from "Variations in the heme oxygenase-1 microsatellite polymorphism are associated with plasma CD14 and viral load in HIV-infected African Americans"

    Article Title: Variations in the heme oxygenase-1 microsatellite polymorphism are associated with plasma CD14 and viral load in HIV-infected African Americans

    Journal: Genes and Immunity

    doi: 10.1038/gene.2011.76

    Heme oxygenase-1 additive GT n repeats negatively correlate with gene expression in PBMCs and CD14 + monocytes from HIV-infected subjects PBMCs from HIV-infected subjects were analyzed for HO-1 transcript analysis. (a) Total mRNA was harvested from thawed PBMCs of HIV viral non-controllers and multiple regression analysis with ethnicity as a covariate yields a significant decline in normalized HO-1 transcript levels as the additive GT n repeats increase (n=34, r= −0.41, β= −3.08, p=0.02). Total mRNA was harvested from thawed PBMCs of HIV viral controllers and multiple regression analysis with ethnicity as a covariate does not lead to a significant decline in normalized HO-1 transcript levels as the additive GT n repeats increase (n=30, r=0.03, β= −0.36, p=0.4). (b) Thawed PBMCs from HAART-suppressed subjects were analyzed by multiparameter flow cytometry for HO-1 expression within CD14 + monocytes. Multiple regression analysis with ethnicity as a covariate yields a significant decline in normalized HO-1 gMFI as the additive GT n repeats increase within CD14 + monocytes (n=25, r= −0.36, β= −30.45, p=0.04). The solid line represents a fitted linear regression line and dashed lines represent the 95% confidence interval band.
    Figure Legend Snippet: Heme oxygenase-1 additive GT n repeats negatively correlate with gene expression in PBMCs and CD14 + monocytes from HIV-infected subjects PBMCs from HIV-infected subjects were analyzed for HO-1 transcript analysis. (a) Total mRNA was harvested from thawed PBMCs of HIV viral non-controllers and multiple regression analysis with ethnicity as a covariate yields a significant decline in normalized HO-1 transcript levels as the additive GT n repeats increase (n=34, r= −0.41, β= −3.08, p=0.02). Total mRNA was harvested from thawed PBMCs of HIV viral controllers and multiple regression analysis with ethnicity as a covariate does not lead to a significant decline in normalized HO-1 transcript levels as the additive GT n repeats increase (n=30, r=0.03, β= −0.36, p=0.4). (b) Thawed PBMCs from HAART-suppressed subjects were analyzed by multiparameter flow cytometry for HO-1 expression within CD14 + monocytes. Multiple regression analysis with ethnicity as a covariate yields a significant decline in normalized HO-1 gMFI as the additive GT n repeats increase within CD14 + monocytes (n=25, r= −0.36, β= −30.45, p=0.04). The solid line represents a fitted linear regression line and dashed lines represent the 95% confidence interval band.

    Techniques Used: Expressing, Infection, Flow Cytometry, Cytometry

    74) Product Images from "Myosin VI regulates gene pairing and transcriptional pause release in T cells"

    Article Title: Myosin VI regulates gene pairing and transcriptional pause release in T cells

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

    doi: 10.1073/pnas.1502461112

    Pairing and repositioning of the LT/TNF locus are dependent on nuclear myosin VI. ( A ) Separation of the homologous LT/TNF alleles was measured in resting or 1-h restimulated Th1 cells in the presence or absence of either TIP or cytochalasin B (CytoB)
    Figure Legend Snippet: Pairing and repositioning of the LT/TNF locus are dependent on nuclear myosin VI. ( A ) Separation of the homologous LT/TNF alleles was measured in resting or 1-h restimulated Th1 cells in the presence or absence of either TIP or cytochalasin B (CytoB)

    Techniques Used:

    Homologous pairing of the LT/TNF alleles correlates with biallelic TNF-α expression in 1-h restimulated Th1 cells. ( A ) Quantitative PCR of TNF-α mRNA expression in differentiating and restimulated Th1 cells. HPRT, hypoxanthine phosphoribosyltransferase.
    Figure Legend Snippet: Homologous pairing of the LT/TNF alleles correlates with biallelic TNF-α expression in 1-h restimulated Th1 cells. ( A ) Quantitative PCR of TNF-α mRNA expression in differentiating and restimulated Th1 cells. HPRT, hypoxanthine phosphoribosyltransferase.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Nuclear Myo VI regulates RNAPII pausing. ( A ) Cumulative fraction plot of PI values for all transcribed genes in resting (dark gray: HR 0) and restimulated (light gray: HR 1) Myo VI-sufficient ( Left ) and Myo VI-deficient ( Right ) Th1 cells. ( B ) Metagene
    Figure Legend Snippet: Nuclear Myo VI regulates RNAPII pausing. ( A ) Cumulative fraction plot of PI values for all transcribed genes in resting (dark gray: HR 0) and restimulated (light gray: HR 1) Myo VI-sufficient ( Left ) and Myo VI-deficient ( Right ) Th1 cells. ( B ) Metagene

    Techniques Used:

    75) Product Images from "The mRNA cap methyltransferase gene TbCMT1 is not essential in vitro but is a virulence factor in vivo for bloodstream form Trypanosoma brucei"

    Article Title: The mRNA cap methyltransferase gene TbCMT1 is not essential in vitro but is a virulence factor in vivo for bloodstream form Trypanosoma brucei

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0201263

    TbCMT1 is not required for T . brucei Lister 427 BSF proliferation in cell culture. (A) Total RNA was purified from TbCMT1 conditional null cells cultured with (+tet) and without tetracycline for 24, 48 and 72 h. TbCMT1 expression was analysed by qRT-PCR normalised to telomerase reverse transcriptase ( TERT ). The delta Ct values (dCt) for nine measurements for each condition are visualised as a swarm plot to show all observations along with representations of the underlying distributions. (B) Cumulative cell counts of triplicate TbCMT1 conditional null mutant cell cultures grown with (plus) and without (minus) tetracycline. (C) Cumulative cell counts of triplicate wild type (WT) and TbCMT1 null cells cultured in parallel. For the data in panels B and C, the cell counts of three biological replicates are reported after 2, 4, 6, 8 and 10 days. The cultures were counted and diluted to 10 5 cells/ml every two days in (B) and to 10 4 cells/ml every two days in (C). The cell counts are reported as the log10 value of the cumulative number of parasites per ml of cell culture allowing for the aforementioned dilution factors.
    Figure Legend Snippet: TbCMT1 is not required for T . brucei Lister 427 BSF proliferation in cell culture. (A) Total RNA was purified from TbCMT1 conditional null cells cultured with (+tet) and without tetracycline for 24, 48 and 72 h. TbCMT1 expression was analysed by qRT-PCR normalised to telomerase reverse transcriptase ( TERT ). The delta Ct values (dCt) for nine measurements for each condition are visualised as a swarm plot to show all observations along with representations of the underlying distributions. (B) Cumulative cell counts of triplicate TbCMT1 conditional null mutant cell cultures grown with (plus) and without (minus) tetracycline. (C) Cumulative cell counts of triplicate wild type (WT) and TbCMT1 null cells cultured in parallel. For the data in panels B and C, the cell counts of three biological replicates are reported after 2, 4, 6, 8 and 10 days. The cultures were counted and diluted to 10 5 cells/ml every two days in (B) and to 10 4 cells/ml every two days in (C). The cell counts are reported as the log10 value of the cumulative number of parasites per ml of cell culture allowing for the aforementioned dilution factors.

    Techniques Used: Cell Culture, Purification, Expressing, Quantitative RT-PCR, Mutagenesis

    Related Articles

    Real-time Polymerase Chain Reaction:

    Article Title: Hormonal regulation of mannan-binding lectin synthesis in hepatocytes
    Article Snippet: .. Assay-on-demand gene expression products from Applied Biosystems were used for quantitative real-time PCR gene expression for MBL2 mRNA (cat. no. Hs01551501_m1) using beta 2 microglobulin (β2m) mRNA (cat. no. Hs99999907_m1) as a housekeeping gene for normalization of the system. .. The amount of cDNA is expressed as the threshold cycle (Ct), which is the number of cycles needed to gain 50% of maximal fluorescence.

    Amplification:

    Article Title: Hormonal regulation of mannan-binding lectin synthesis in hepatocytes
    Article Snippet: For the measurement of mRNA contents, total RNA was extracted from cells with the RNeasy mini kit (Qiagen) followed by DNAse treatment with deoxyribonuclease I, amplification grade (Invitrogen). .. Assay-on-demand gene expression products from Applied Biosystems were used for quantitative real-time PCR gene expression for MBL2 mRNA (cat. no. Hs01551501_m1) using beta 2 microglobulin (β2m) mRNA (cat. no. Hs99999907_m1) as a housekeeping gene for normalization of the system.

    Fluorescence:

    Article Title: Hormonal regulation of mannan-binding lectin synthesis in hepatocytes
    Article Snippet: Assay-on-demand gene expression products from Applied Biosystems were used for quantitative real-time PCR gene expression for MBL2 mRNA (cat. no. Hs01551501_m1) using beta 2 microglobulin (β2m) mRNA (cat. no. Hs99999907_m1) as a housekeeping gene for normalization of the system. .. The amount of cDNA is expressed as the threshold cycle (Ct), which is the number of cycles needed to gain 50% of maximal fluorescence.

    Purification:

    Article Title: Hormonal regulation of mannan-binding lectin synthesis in hepatocytes
    Article Snippet: Assay-on-demand gene expression products from Applied Biosystems were used for quantitative real-time PCR gene expression for MBL2 mRNA (cat. no. Hs01551501_m1) using beta 2 microglobulin (β2m) mRNA (cat. no. Hs99999907_m1) as a housekeeping gene for normalization of the system. .. We found this approach suboptimal due to the highly purified nature of the plasmid compared to cDNA produced from the cells.

    Produced:

    Article Title: Hormonal regulation of mannan-binding lectin synthesis in hepatocytes
    Article Snippet: The MBL2 mRNA expression levels were quantified using a two-step RT–PCR based on detection of a fluorescent signal produced proportionally during the amplification of a PCR product, employing Taq Man chemistry and the ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). .. Assay-on-demand gene expression products from Applied Biosystems were used for quantitative real-time PCR gene expression for MBL2 mRNA (cat. no. Hs01551501_m1) using beta 2 microglobulin (β2m) mRNA (cat. no. Hs99999907_m1) as a housekeeping gene for normalization of the system.

    Polymerase Chain Reaction:

    Article Title: Hormonal regulation of mannan-binding lectin synthesis in hepatocytes
    Article Snippet: The MBL2 mRNA expression levels were quantified using a two-step RT–PCR based on detection of a fluorescent signal produced proportionally during the amplification of a PCR product, employing Taq Man chemistry and the ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). .. Assay-on-demand gene expression products from Applied Biosystems were used for quantitative real-time PCR gene expression for MBL2 mRNA (cat. no. Hs01551501_m1) using beta 2 microglobulin (β2m) mRNA (cat. no. Hs99999907_m1) as a housekeeping gene for normalization of the system.

    Expressing:

    Article Title: Hormonal regulation of mannan-binding lectin synthesis in hepatocytes
    Article Snippet: .. Assay-on-demand gene expression products from Applied Biosystems were used for quantitative real-time PCR gene expression for MBL2 mRNA (cat. no. Hs01551501_m1) using beta 2 microglobulin (β2m) mRNA (cat. no. Hs99999907_m1) as a housekeeping gene for normalization of the system. .. The amount of cDNA is expressed as the threshold cycle (Ct), which is the number of cycles needed to gain 50% of maximal fluorescence.

    Sequencing:

    Article Title: Hormonal regulation of mannan-binding lectin synthesis in hepatocytes
    Article Snippet: The MBL2 mRNA expression levels were quantified using a two-step RT–PCR based on detection of a fluorescent signal produced proportionally during the amplification of a PCR product, employing Taq Man chemistry and the ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). .. Assay-on-demand gene expression products from Applied Biosystems were used for quantitative real-time PCR gene expression for MBL2 mRNA (cat. no. Hs01551501_m1) using beta 2 microglobulin (β2m) mRNA (cat. no. Hs99999907_m1) as a housekeeping gene for normalization of the system.

    Plasmid Preparation:

    Article Title: Hormonal regulation of mannan-binding lectin synthesis in hepatocytes
    Article Snippet: Assay-on-demand gene expression products from Applied Biosystems were used for quantitative real-time PCR gene expression for MBL2 mRNA (cat. no. Hs01551501_m1) using beta 2 microglobulin (β2m) mRNA (cat. no. Hs99999907_m1) as a housekeeping gene for normalization of the system. .. To express the production of MBL2 mRNA as the number of copies, we initially calibrated against a plasmid containing inserted MBL2 cDNA.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Hormonal regulation of mannan-binding lectin synthesis in hepatocytes
    Article Snippet: Paragraph title: Quantitative real-time reverse transcriptase–polymerase chain reaction (RT–PCR) ... Assay-on-demand gene expression products from Applied Biosystems were used for quantitative real-time PCR gene expression for MBL2 mRNA (cat. no. Hs01551501_m1) using beta 2 microglobulin (β2m) mRNA (cat. no. Hs99999907_m1) as a housekeeping gene for normalization of the system.

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