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Functional characterization of early and late passage <t>SBK.</t> (A-B) <t>RNA-seq</t> analysis was conducted on early and late passage SBK stimulated with IL-17 and IFN-γ for 48 hours. ANOVA analysis was performed, and the resulting -log 10 (adjusted p-value for false discovery) vs. log 2 fold-change for difference of treatment was plotted. Values with | log 2 fold-change | > 2 and an adjusted p value
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1) Product Images from "Prolonging culture of primary human keratinocytes isolated from suction blisters with the Rho kinase inhibitor Y-27632"

Article Title: Prolonging culture of primary human keratinocytes isolated from suction blisters with the Rho kinase inhibitor Y-27632

Journal: PLoS ONE

doi: 10.1371/journal.pone.0198862

Functional characterization of early and late passage SBK. (A-B) RNA-seq analysis was conducted on early and late passage SBK stimulated with IL-17 and IFN-γ for 48 hours. ANOVA analysis was performed, and the resulting -log 10 (adjusted p-value for false discovery) vs. log 2 fold-change for difference of treatment was plotted. Values with | log 2 fold-change | > 2 and an adjusted p value
Figure Legend Snippet: Functional characterization of early and late passage SBK. (A-B) RNA-seq analysis was conducted on early and late passage SBK stimulated with IL-17 and IFN-γ for 48 hours. ANOVA analysis was performed, and the resulting -log 10 (adjusted p-value for false discovery) vs. log 2 fold-change for difference of treatment was plotted. Values with | log 2 fold-change | > 2 and an adjusted p value

Techniques Used: Functional Assay, RNA Sequencing Assay

Y-27632 delays terminal differentiation in SBK. RNA-seq analysis was performed on RNA isolated from SBK grown in the absence (-Y; passages 4, 5, and 6) and presence (+Y; passages 5 and 6) of Y-27632. (A) A plot of the log 2 least square mean of all the genes from each culture condition. The dotted lines indicate genes with greater than 2.6 fold change; the red dots indicate the subset of those with an adjusted p value
Figure Legend Snippet: Y-27632 delays terminal differentiation in SBK. RNA-seq analysis was performed on RNA isolated from SBK grown in the absence (-Y; passages 4, 5, and 6) and presence (+Y; passages 5 and 6) of Y-27632. (A) A plot of the log 2 least square mean of all the genes from each culture condition. The dotted lines indicate genes with greater than 2.6 fold change; the red dots indicate the subset of those with an adjusted p value

Techniques Used: RNA Sequencing Assay, Isolation

2) Product Images from "Transcriptomes of Trypanosoma brucei rhodesiense from sleeping sickness patients, rodents and culture: Effects of strain, growth conditions and RNA preparation methods"

Article Title: Transcriptomes of Trypanosoma brucei rhodesiense from sleeping sickness patients, rodents and culture: Effects of strain, growth conditions and RNA preparation methods

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0006280

Poly(A) selection and rRNA depletion: effect of mRNA length. Different datasets were compared using DeSeq2 ( S2 Table Sheet 1). The ratios of rRNA-depleted divided by poly(A)+ are shown on the y axis, and the mRNA length on the x-axis. Log-transformed values were used for the graphs and the regression analysis, but for clarity, the mRNA length axis has been labelled with the non log-transformed values. A. Results for all pooled datasets. Differences in RNA abundance were classed as significant if the adjusted p-value was less than 0.01, and the magnitude of the difference was at least 2-fold [ 64 ]. Correlation coefficients were calculated by Microsoft Excel. B. Results for selected cultures (the ones with values for most full genes). C. As (B), but with mRNAs encoding protein kinases in orange and mRNAs encoding ribosomal proteins in green. D. As (B), but with mRNAs encoding RNA-binding proteins in pink.
Figure Legend Snippet: Poly(A) selection and rRNA depletion: effect of mRNA length. Different datasets were compared using DeSeq2 ( S2 Table Sheet 1). The ratios of rRNA-depleted divided by poly(A)+ are shown on the y axis, and the mRNA length on the x-axis. Log-transformed values were used for the graphs and the regression analysis, but for clarity, the mRNA length axis has been labelled with the non log-transformed values. A. Results for all pooled datasets. Differences in RNA abundance were classed as significant if the adjusted p-value was less than 0.01, and the magnitude of the difference was at least 2-fold [ 64 ]. Correlation coefficients were calculated by Microsoft Excel. B. Results for selected cultures (the ones with values for most full genes). C. As (B), but with mRNAs encoding protein kinases in orange and mRNAs encoding ribosomal proteins in green. D. As (B), but with mRNAs encoding RNA-binding proteins in pink.

Techniques Used: Selection, Transformation Assay, RNA Binding Assay

Reporter experiments: Effect of mRNA length on poly(A) selection and mRNA abundance. A. Cartoon of an intact Tb927.4.1500 locus (above) and a locus with integrated PAC-GFP (not to scale). B. Structures of GFP fusion mRNAs (to scale) with lengths. The codon optimality scores are calculated using a formula provided by Prof. M. Carrington (Cambridge University, personal communication). Approximate half-lives were estimated by real-time PCR of mRNA prepared 30 min after addition of Actinomycin D; each clone was measured once. C. Sample Northern blot for Tb927.4.1500; other blots are in S4D Fig . Amounts of GFP signal or PCR product relative to the result for the 1.3 kb mRNA. The boxes indicate the median value with 25th and 75th percentiles; whiskers extend to the most extreme data point that is no more than 1.5 times the length of the box away from the box. Tiny circles are outliers. E. Sample Northern blot for Tb927.8.1050; other blots are in S3F Fig . Amounts of GFP signal or PCR product relative to the result for the 2.7 kb mRNA. If less than 4 measurements were available, individual results are shown as coloured circles. G. Ratio of poly(A)+ to total mRNA. To make the values for the two genes comparable, the abundances of the Tb927.4.1500 reporter mRNAs were calculated relative to the 2.2 kb mRNA. (This results in exclusion of one dataset that lacks a value for that length.) The average relative mRNA abundance in poly(A)+ was divided by the relative abundance in total RNA. Results using medians were similar. The exponential curve was calculated in Kaleidograph using the combined data from both genes.
Figure Legend Snippet: Reporter experiments: Effect of mRNA length on poly(A) selection and mRNA abundance. A. Cartoon of an intact Tb927.4.1500 locus (above) and a locus with integrated PAC-GFP (not to scale). B. Structures of GFP fusion mRNAs (to scale) with lengths. The codon optimality scores are calculated using a formula provided by Prof. M. Carrington (Cambridge University, personal communication). Approximate half-lives were estimated by real-time PCR of mRNA prepared 30 min after addition of Actinomycin D; each clone was measured once. C. Sample Northern blot for Tb927.4.1500; other blots are in S4D Fig . Amounts of GFP signal or PCR product relative to the result for the 1.3 kb mRNA. The boxes indicate the median value with 25th and 75th percentiles; whiskers extend to the most extreme data point that is no more than 1.5 times the length of the box away from the box. Tiny circles are outliers. E. Sample Northern blot for Tb927.8.1050; other blots are in S3F Fig . Amounts of GFP signal or PCR product relative to the result for the 2.7 kb mRNA. If less than 4 measurements were available, individual results are shown as coloured circles. G. Ratio of poly(A)+ to total mRNA. To make the values for the two genes comparable, the abundances of the Tb927.4.1500 reporter mRNAs were calculated relative to the 2.2 kb mRNA. (This results in exclusion of one dataset that lacks a value for that length.) The average relative mRNA abundance in poly(A)+ was divided by the relative abundance in total RNA. Results using medians were similar. The exponential curve was calculated in Kaleidograph using the combined data from both genes.

Techniques Used: Selection, Real-time Polymerase Chain Reaction, Northern Blot, Polymerase Chain Reaction

Relationships between cell density and expression of chosen genes. A. Principal component analysis including blood and CSF RNAs prepared using a common protocol, and for rRNA-depleted RNA from culture. B. The mRNAs that were significantly different between human blood and CSF were categorised according to whether they show peak expression in a particular cell cycle stage. RNAs that show no cell cycle regulation are not included. C. Expression of EIF4A mRNA (RPM) relative to cell density (log scale). The key is on the blot and sample numbers are indicated. Note that the densities for the human blood samples are approximate because they were estimated from stained thin films (see methods ). D. As (C), but showing PIP39 mRNA. E. As (C), but showing COX IX mRNA. Additional results are in S6 Fig .
Figure Legend Snippet: Relationships between cell density and expression of chosen genes. A. Principal component analysis including blood and CSF RNAs prepared using a common protocol, and for rRNA-depleted RNA from culture. B. The mRNAs that were significantly different between human blood and CSF were categorised according to whether they show peak expression in a particular cell cycle stage. RNAs that show no cell cycle regulation are not included. C. Expression of EIF4A mRNA (RPM) relative to cell density (log scale). The key is on the blot and sample numbers are indicated. Note that the densities for the human blood samples are approximate because they were estimated from stained thin films (see methods ). D. As (C), but showing PIP39 mRNA. E. As (C), but showing COX IX mRNA. Additional results are in S6 Fig .

Techniques Used: Expressing, Staining

3) Product Images from "miRNA in Plasma Exosome is Stable under Different Storage Conditions"

Article Title: miRNA in Plasma Exosome is Stable under Different Storage Conditions

Journal: Molecules

doi: 10.3390/molecules19021568

Concentration of RNA extracted from exosomes and plasma under different conditions.
Figure Legend Snippet: Concentration of RNA extracted from exosomes and plasma under different conditions.

Techniques Used: Concentration Assay

4) Product Images from "Sensitivity to splicing modulation of BCL2 family genes defines cancer therapeutic strategies for splicing modulators"

Article Title: Sensitivity to splicing modulation of BCL2 family genes defines cancer therapeutic strategies for splicing modulators

Journal: Nature Communications

doi: 10.1038/s41467-018-08150-5

E7107 induces BCL2A1-dependent apoptosis in melanoma cell lines. a RT quantitative PCR (RT-qPCR) analysis of total mRNA levels (pan), exonic mRNA levels covering the junction of exons (exon), and pre-mRNA levels detecting the intron (intron) of BCL2A1 . HT144 cells were pre-treated with 100 µg ml −1 cycloheximide (CHX) for 1 h, followed by addition of E7107 as indicated for 12 h to inhibit nonsense-mediated mRNA decay (NMD) before collecting RNA samples. Schematic representations of TaqMan primers and probes were shown. Boxes: exons; gray lines: introns; arrows: primers; and black lines: probes. b Box plots showing the distribution of E7107 Emax in the solid tumor cell lines separated by tissue/lineage: melanoma ( n = 24), breast ( n = 33), colon ( n = 27), kidney ( n = 10), lung ( n = 91), and pancreas ( n = 24). The box plots exhibit five number summary from bottom to top: minimum, first quartile, median, third quartile, and maximum/outliers. c Heatmap of BCL2A1 mRNA expression (based on CCLE RNA-seq, sorted from high to low in the solid tumor cell lines separated by tissue/lineage) and E7107 Emax. d RT-qPCR analysis of BCL2A1 mRNA levels (exon1/2 junction) in vector control (Vector) or stable BCL2A1 cDNA-expressing (BCL2A1) melanoma cell lines. Data represent means ± SD of biological triplicates. e Growth curves measured by CellTiter-Glo (CTG) and f apoptosis induction curves measured by IncuCyte Caspase-3/7 Green Apoptosis Assay in melanoma cell lines stably transduced with vector control (empty vector) or BCL2A1 cDNA (BCL2A1 cDNA) treated with E7107 for 72 h. Data represent means ± SD of biological triplicates
Figure Legend Snippet: E7107 induces BCL2A1-dependent apoptosis in melanoma cell lines. a RT quantitative PCR (RT-qPCR) analysis of total mRNA levels (pan), exonic mRNA levels covering the junction of exons (exon), and pre-mRNA levels detecting the intron (intron) of BCL2A1 . HT144 cells were pre-treated with 100 µg ml −1 cycloheximide (CHX) for 1 h, followed by addition of E7107 as indicated for 12 h to inhibit nonsense-mediated mRNA decay (NMD) before collecting RNA samples. Schematic representations of TaqMan primers and probes were shown. Boxes: exons; gray lines: introns; arrows: primers; and black lines: probes. b Box plots showing the distribution of E7107 Emax in the solid tumor cell lines separated by tissue/lineage: melanoma ( n = 24), breast ( n = 33), colon ( n = 27), kidney ( n = 10), lung ( n = 91), and pancreas ( n = 24). The box plots exhibit five number summary from bottom to top: minimum, first quartile, median, third quartile, and maximum/outliers. c Heatmap of BCL2A1 mRNA expression (based on CCLE RNA-seq, sorted from high to low in the solid tumor cell lines separated by tissue/lineage) and E7107 Emax. d RT-qPCR analysis of BCL2A1 mRNA levels (exon1/2 junction) in vector control (Vector) or stable BCL2A1 cDNA-expressing (BCL2A1) melanoma cell lines. Data represent means ± SD of biological triplicates. e Growth curves measured by CellTiter-Glo (CTG) and f apoptosis induction curves measured by IncuCyte Caspase-3/7 Green Apoptosis Assay in melanoma cell lines stably transduced with vector control (empty vector) or BCL2A1 cDNA (BCL2A1 cDNA) treated with E7107 for 72 h. Data represent means ± SD of biological triplicates

Techniques Used: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, RNA Sequencing Assay, Plasmid Preparation, CTG Assay, Apoptosis Assay, Stable Transfection, Transduction

5) Product Images from "H19 potentiates let-7 family expression through reducing PTBP1 binding to their precursors in cholestasis"

Article Title: H19 potentiates let-7 family expression through reducing PTBP1 binding to their precursors in cholestasis

Journal: Cell Death & Disease

doi: 10.1038/s41419-019-1423-6

H19 increases mature let-7a-5p/7d-5p/7f-5p levels in mouse bile duct ligation (BDL) model. a The heat map showing differential hepatic expression of let-7 family members. Wild-type (WT) mice were injected with either an empty adeno-associated viral vector serotype 8 (AAV8) virus (Null) or an H19 -expression AAV8 virus (H19) for 1 month to achieve stable gene expression. Then, these mice were subjected to either sham or BDL surgery for 1 week. Livers were then collected and RNA was extracted and subjected to microRNA-sequencing (miR-seq). The average count numbers of top 32 miRNAs in each group were plotted. b The count numbers of individual let-7 family members from the miR-seq results were graphed. c Quantitative PCR (qPCR) of let-7a-5p, let-7d-5p, and let-7f-5p in Null-BDL and H19-BDL mouse livers ( n = 5 mice/group). d qPCR of let-7a-5p, let-7d-5p, and let-7f-5p in BDL-operated paternal H19 knockout (con) and maternal H19 knockout (HKO) mouse livers ( n = 5 mice/group). Data were presented as the mean ± SEM from triplicate assays. * P
Figure Legend Snippet: H19 increases mature let-7a-5p/7d-5p/7f-5p levels in mouse bile duct ligation (BDL) model. a The heat map showing differential hepatic expression of let-7 family members. Wild-type (WT) mice were injected with either an empty adeno-associated viral vector serotype 8 (AAV8) virus (Null) or an H19 -expression AAV8 virus (H19) for 1 month to achieve stable gene expression. Then, these mice were subjected to either sham or BDL surgery for 1 week. Livers were then collected and RNA was extracted and subjected to microRNA-sequencing (miR-seq). The average count numbers of top 32 miRNAs in each group were plotted. b The count numbers of individual let-7 family members from the miR-seq results were graphed. c Quantitative PCR (qPCR) of let-7a-5p, let-7d-5p, and let-7f-5p in Null-BDL and H19-BDL mouse livers ( n = 5 mice/group). d qPCR of let-7a-5p, let-7d-5p, and let-7f-5p in BDL-operated paternal H19 knockout (con) and maternal H19 knockout (HKO) mouse livers ( n = 5 mice/group). Data were presented as the mean ± SEM from triplicate assays. * P

Techniques Used: Ligation, Expressing, Mouse Assay, Injection, Plasmid Preparation, Sequencing, Real-time Polymerase Chain Reaction, Knock-Out

6) Product Images from "4-O-Methylhonokiol Influences Normal Cardiovascular Development in Medaka Embryo"

Article Title: 4-O-Methylhonokiol Influences Normal Cardiovascular Development in Medaka Embryo

Journal: Molecules

doi: 10.3390/molecules24030475

Embryos treated with MH lead to altered cardiac biomarker gene expression and altered expression of genes involved in thrombosis, fibrinolysis, and vascular tone. Real-time RT-qPCR was performed on total RNA isolated from each group of embryos using described primers in Table 1 leading to the amplification of the target gene. ( A ) RT-qPCR analysis of factor XI (FXI), factor VII (FVII), factor X (FX), plasminogen activator inhibitor 1 (PAI-1), tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), endothelin B (ETB), and angiotensin type 1 receptor associated protein (ATRAP) isolated from control and MH-treated embryos. ( B ) RT-qPCR analysis of natriuretic peptide A, Troponin T, ErbB3, and Nrg2. Data were normalized to the eukaryotic elongation factor 1-alpha (eEf1α) polymerase chain reaction signal. Each value represents mean ± SEM ( n = 100, replicated four times). * indicates a value is significant versus the respective control group ( p
Figure Legend Snippet: Embryos treated with MH lead to altered cardiac biomarker gene expression and altered expression of genes involved in thrombosis, fibrinolysis, and vascular tone. Real-time RT-qPCR was performed on total RNA isolated from each group of embryos using described primers in Table 1 leading to the amplification of the target gene. ( A ) RT-qPCR analysis of factor XI (FXI), factor VII (FVII), factor X (FX), plasminogen activator inhibitor 1 (PAI-1), tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), endothelin B (ETB), and angiotensin type 1 receptor associated protein (ATRAP) isolated from control and MH-treated embryos. ( B ) RT-qPCR analysis of natriuretic peptide A, Troponin T, ErbB3, and Nrg2. Data were normalized to the eukaryotic elongation factor 1-alpha (eEf1α) polymerase chain reaction signal. Each value represents mean ± SEM ( n = 100, replicated four times). * indicates a value is significant versus the respective control group ( p

Techniques Used: Biomarker Assay, Expressing, Quantitative RT-PCR, Isolation, Amplification, Polymerase Chain Reaction

Embryos treated with MH lead to altered expression of genes involved in cell regulation, oxidative stress, and inflammation. Real-time RT-qPCR was performed on total RNA isolated from each group of embryos using described primers in Table 1 leading to the amplification of the target gene. RT-qPCR analysis of FoxO1, catalase, glutathione peroxidase (GPX2), glutathione-s-transferase (GSTA), superoxide dismutase 2 (SOD-2), Interleukin 1 beta (IL-1β), and tissue necrosis factor-alpha (TNF-α) isolated from control and MH-treated embryos. Data were normalized to the eEf1α polymerase chain reaction signal. Each value represents mean ± SEM ( n = 100, replicated four times). Statistical analysis was performed by two-way ANOVA followed by post-hoc Bonferroni test. * indicates values whic h are significant versus the respective control group ( p
Figure Legend Snippet: Embryos treated with MH lead to altered expression of genes involved in cell regulation, oxidative stress, and inflammation. Real-time RT-qPCR was performed on total RNA isolated from each group of embryos using described primers in Table 1 leading to the amplification of the target gene. RT-qPCR analysis of FoxO1, catalase, glutathione peroxidase (GPX2), glutathione-s-transferase (GSTA), superoxide dismutase 2 (SOD-2), Interleukin 1 beta (IL-1β), and tissue necrosis factor-alpha (TNF-α) isolated from control and MH-treated embryos. Data were normalized to the eEf1α polymerase chain reaction signal. Each value represents mean ± SEM ( n = 100, replicated four times). Statistical analysis was performed by two-way ANOVA followed by post-hoc Bonferroni test. * indicates values whic h are significant versus the respective control group ( p

Techniques Used: Expressing, Quantitative RT-PCR, Isolation, Amplification, Polymerase Chain Reaction

MH controls the Wnt signal transduction pathway, a main regulator of development. Real-time RT-qPCR was performed on total RNA isolated from each group of embryos using described primers in Table 1 , leading to the amplification of the target gene. RT-qPCR analysis of Wnt 1, transforming growth factor beta 2 (TGF-β2), frizzled 2 (Fzd2), low-density lipoprotein receptor-related protein 5 (LRP5), dishevelled (Dvl), glycogen synthase kinase 3 beta (GSK-3β), β-catenin, and dickkopf 1 (DKK1) isolated from the control and MH-treated embryos. Each value represents mean ± SEM ( n = 100, replicated four times). Statistical analysis was performed by two-way ANOVA followed by post-hoc Bonferroni test. * indicates values which are significant versus the respective control group ( p
Figure Legend Snippet: MH controls the Wnt signal transduction pathway, a main regulator of development. Real-time RT-qPCR was performed on total RNA isolated from each group of embryos using described primers in Table 1 , leading to the amplification of the target gene. RT-qPCR analysis of Wnt 1, transforming growth factor beta 2 (TGF-β2), frizzled 2 (Fzd2), low-density lipoprotein receptor-related protein 5 (LRP5), dishevelled (Dvl), glycogen synthase kinase 3 beta (GSK-3β), β-catenin, and dickkopf 1 (DKK1) isolated from the control and MH-treated embryos. Each value represents mean ± SEM ( n = 100, replicated four times). Statistical analysis was performed by two-way ANOVA followed by post-hoc Bonferroni test. * indicates values which are significant versus the respective control group ( p

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

7) Product Images from "How Rainforest Conversion to Agricultural Systems in Sumatra (Indonesia) Affects Active Soil Bacterial Communities"

Article Title: How Rainforest Conversion to Agricultural Systems in Sumatra (Indonesia) Affects Active Soil Bacterial Communities

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.02381

Community composition of active soil bacterial communities in three different land use systems and rainforest reference sites in two different landscapes. All plots were clustered according to their respective land use and landscape. Community compositions are displayed as relative abundances at order level based on 16S rRNA sequences obtained from extracted soil RNA. Taxa with abundances below 1% in each land use system were summarized as “rare taxa.” The detected orders are grouped to corresponding phylum (for details see Supplementary Table S2 ).
Figure Legend Snippet: Community composition of active soil bacterial communities in three different land use systems and rainforest reference sites in two different landscapes. All plots were clustered according to their respective land use and landscape. Community compositions are displayed as relative abundances at order level based on 16S rRNA sequences obtained from extracted soil RNA. Taxa with abundances below 1% in each land use system were summarized as “rare taxa.” The detected orders are grouped to corresponding phylum (for details see Supplementary Table S2 ).

Techniques Used:

8) Product Images from "The acidic domain of the hepatitis C virus NS4A protein is required for viral assembly and envelopment through interactions with the viral E1 glycoprotein"

Article Title: The acidic domain of the hepatitis C virus NS4A protein is required for viral assembly and envelopment through interactions with the viral E1 glycoprotein

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1007163

The NS4A Y45F mutation results in production of partially formed virions. Supernatants harvested from Huh7.5 cells 48 hours post-electroporation of NS4A WT, NS4A Y45F, or ΔE1/E2 in vitro transcribed HCV RNA (JFH1) were analyzed for HCV RNA by RT-qPCR (A) or Core protein by ELISA (B). Data in A are presented as mean ± SEM (n = 3) and analyzed by unpaired t-test. ****P
Figure Legend Snippet: The NS4A Y45F mutation results in production of partially formed virions. Supernatants harvested from Huh7.5 cells 48 hours post-electroporation of NS4A WT, NS4A Y45F, or ΔE1/E2 in vitro transcribed HCV RNA (JFH1) were analyzed for HCV RNA by RT-qPCR (A) or Core protein by ELISA (B). Data in A are presented as mean ± SEM (n = 3) and analyzed by unpaired t-test. ****P

Techniques Used: Mutagenesis, Electroporation, In Vitro, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

9) Product Images from "Epigallocatechin-3-gallate local pre-exposure application prevents SHIV rectal infection of macaques"

Article Title: Epigallocatechin-3-gallate local pre-exposure application prevents SHIV rectal infection of macaques

Journal: Mucosal immunology

doi: 10.1038/s41385-018-0025-4

EGCG protects macaques from intra-rectal SHIV infection. (a) Experimental design of EGCG protective effect on macaques. Sixteen male macaques were administrated with 2 ml of 5 mM EGCG (8 animals) or 2 ml of PBS (8 animals) atraumatically in rectum 10 min prior to each SHIV SF162P3N challenge. All animals were rectally inoculated with SHIV SF162P3N (10 TCID 50 ) for up to 8 times or until infection occurred. All animals were biopsied at week 20 and necropsied at week 36 postinfection for the evaluation of SHIV RNA and proviral DNA in the multiple tissues. (b, c) Longitudinal assessment of the plasma SHIV RNA (copies ml -1 ) levels in the animals with intrarectal pretreatment with PBS (control) or EGCG prior to SHIV challenges (up to 8 times or till infection occurred). Duplicate plasma samples were analyzed for SHIV RNA detection. Animals were considered infected and the virus challenges were stopped following two consecutive positive plasma SHIV RNA results.
Figure Legend Snippet: EGCG protects macaques from intra-rectal SHIV infection. (a) Experimental design of EGCG protective effect on macaques. Sixteen male macaques were administrated with 2 ml of 5 mM EGCG (8 animals) or 2 ml of PBS (8 animals) atraumatically in rectum 10 min prior to each SHIV SF162P3N challenge. All animals were rectally inoculated with SHIV SF162P3N (10 TCID 50 ) for up to 8 times or until infection occurred. All animals were biopsied at week 20 and necropsied at week 36 postinfection for the evaluation of SHIV RNA and proviral DNA in the multiple tissues. (b, c) Longitudinal assessment of the plasma SHIV RNA (copies ml -1 ) levels in the animals with intrarectal pretreatment with PBS (control) or EGCG prior to SHIV challenges (up to 8 times or till infection occurred). Duplicate plasma samples were analyzed for SHIV RNA detection. Animals were considered infected and the virus challenges were stopped following two consecutive positive plasma SHIV RNA results.

Techniques Used: Infection, RNA Detection

SHIV RNA and DNA detection in multiple tissues necropsied at week 36 post first SHIV challenge. SHIV RNA (a) and DNA (b) assays in the indicated tissues from the animals of PBS control (red symbols, n=6∼8) and EGCG group (green symbols, n=8) at necropsy. Log SHIV copies 2 μg -1 total genomic RNA or DNA equivalents are shown. Symbols represent individual animals and are pooled from three independent experiments. Triplication tissue samples were conducted in each independent experiment. The dot line: the detection threshold. GI: gastrointestinal tract; LN: lymph nodes; Jej-Mes: jejunal mesenteric; Col-Mes: colonal mesenteric; IEL: intraepithelial lymphocytes.
Figure Legend Snippet: SHIV RNA and DNA detection in multiple tissues necropsied at week 36 post first SHIV challenge. SHIV RNA (a) and DNA (b) assays in the indicated tissues from the animals of PBS control (red symbols, n=6∼8) and EGCG group (green symbols, n=8) at necropsy. Log SHIV copies 2 μg -1 total genomic RNA or DNA equivalents are shown. Symbols represent individual animals and are pooled from three independent experiments. Triplication tissue samples were conducted in each independent experiment. The dot line: the detection threshold. GI: gastrointestinal tract; LN: lymph nodes; Jej-Mes: jejunal mesenteric; Col-Mes: colonal mesenteric; IEL: intraepithelial lymphocytes.

Techniques Used:

EGCG inhibits viral infectivity of a broad spectrum of AIDS-related viruses. (a) TZM-bl cells were treated with the indicated concentrations of green tea-derived catechins (EC, EGC, ECG and EGCG) for 10 min prior to infection with different strains of HIV-1 (Bal, NL4-3), SHIV (SF162P3N, KU-1), or SIV (mac239, mac251). Viral infectivity was assessed by luciferase activity, which is expressed as a percentage relative to that of the control (untreated). The half maximal inhibitory concentration (IC 50 ) of EGCG is indicated, which was calculated based on the untreated control by the method of Reed and Muench. (b) Human peripheral blood monocyte-derived macrophages were incubated with the indicated doses of EGCG for 10 min prior to HIV-1 Bal infection. Culture supernatant was collected on day 7 post-infection for HIV-1 reverse transcriptase (RT) assay. Cellular RNA was subjected to the real time RT-PCR for HIV-1 gag and GAPDH RNA. Data are expressed as HIV-1 RNA levels relative (%) to untreated control, which is defined as 100%. (c) Primary lymphocytes and macrophages from rhesus macaques were treated with or without EGCG (50 μM) for 10 min prior to SHIVSF162P3N infection. Intracellular gag RNA was measured by the real time PCR at day 5 post infection. Data are shown as mean ± SD, representative of three independent experiments with 3-4 replicates. * P
Figure Legend Snippet: EGCG inhibits viral infectivity of a broad spectrum of AIDS-related viruses. (a) TZM-bl cells were treated with the indicated concentrations of green tea-derived catechins (EC, EGC, ECG and EGCG) for 10 min prior to infection with different strains of HIV-1 (Bal, NL4-3), SHIV (SF162P3N, KU-1), or SIV (mac239, mac251). Viral infectivity was assessed by luciferase activity, which is expressed as a percentage relative to that of the control (untreated). The half maximal inhibitory concentration (IC 50 ) of EGCG is indicated, which was calculated based on the untreated control by the method of Reed and Muench. (b) Human peripheral blood monocyte-derived macrophages were incubated with the indicated doses of EGCG for 10 min prior to HIV-1 Bal infection. Culture supernatant was collected on day 7 post-infection for HIV-1 reverse transcriptase (RT) assay. Cellular RNA was subjected to the real time RT-PCR for HIV-1 gag and GAPDH RNA. Data are expressed as HIV-1 RNA levels relative (%) to untreated control, which is defined as 100%. (c) Primary lymphocytes and macrophages from rhesus macaques were treated with or without EGCG (50 μM) for 10 min prior to SHIVSF162P3N infection. Intracellular gag RNA was measured by the real time PCR at day 5 post infection. Data are shown as mean ± SD, representative of three independent experiments with 3-4 replicates. * P

Techniques Used: Infection, Derivative Assay, Luciferase, Activity Assay, Concentration Assay, Incubation, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

10) Product Images from "Identification of phloem-associated translatome alterations during leaf development in Prunus domestica L."

Article Title: Identification of phloem-associated translatome alterations during leaf development in Prunus domestica L.

Journal: Horticulture Research

doi: 10.1038/s41438-018-0092-4

Isolation of translatome RNA. a Representative Agilent 2100 bioanalyzer gel-like image showing RNA isolated after incubation with Anti-FLAG magnetic beads taken from the unbound (blue labels) or bound (orange labels) fraction. High-quality RNA was successfully recovered in the bound fraction for all translatome HF-RPL18 plant lines, but not from non-transgenic controls. b Quantification of RNA recovered from the bound fraction. RNA was measured on a NanoDrop 2000 and is shown in nanograms. Bars represent the mean of four biological replicates ± standard error. c Relative HF-RPL18 transcript accumulation detected in total RNA (white bars) and translating mRNA samples (orange bars). Bars represent the mean of three biological replicates ± standard error. All samples are normalized to p35S total RNA
Figure Legend Snippet: Isolation of translatome RNA. a Representative Agilent 2100 bioanalyzer gel-like image showing RNA isolated after incubation with Anti-FLAG magnetic beads taken from the unbound (blue labels) or bound (orange labels) fraction. High-quality RNA was successfully recovered in the bound fraction for all translatome HF-RPL18 plant lines, but not from non-transgenic controls. b Quantification of RNA recovered from the bound fraction. RNA was measured on a NanoDrop 2000 and is shown in nanograms. Bars represent the mean of four biological replicates ± standard error. c Relative HF-RPL18 transcript accumulation detected in total RNA (white bars) and translating mRNA samples (orange bars). Bars represent the mean of three biological replicates ± standard error. All samples are normalized to p35S total RNA

Techniques Used: Isolation, Incubation, Magnetic Beads, Transgenic Assay

11) Product Images from "Heterologous Expression of Serine Hydroxymethyltransferase-3 From Rice Confers Tolerance to Salinity Stress in E. coli and Arabidopsis"

Article Title: Heterologous Expression of Serine Hydroxymethyltransferase-3 From Rice Confers Tolerance to Salinity Stress in E. coli and Arabidopsis

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2019.00217

Effect of salt stress on the gene expression profiling of wild-type and Arabidopsis overexpressing OsSHMT3 . Seedlings (14-day-old), subjected to 100 mM NaCl stress for 24 h, were used for RNA extraction. Venn diagram showing the genes in wild-type and OE5 that are (A) up-regulated, and (B) down-regulated. (C) Seedlings (14-day-old) of the wild-type, OE3 and OE5 were subjected to NaCl (100 mM) stress for 24 h, and the whole seedlings were used for assaying the relative expression of the salt stress responsive genes. Actin was used as an internal control. Values ( n = 9) are mean ± SE and different letters on the histograms indicate that the values differ significantly (two-way ANOVA; P
Figure Legend Snippet: Effect of salt stress on the gene expression profiling of wild-type and Arabidopsis overexpressing OsSHMT3 . Seedlings (14-day-old), subjected to 100 mM NaCl stress for 24 h, were used for RNA extraction. Venn diagram showing the genes in wild-type and OE5 that are (A) up-regulated, and (B) down-regulated. (C) Seedlings (14-day-old) of the wild-type, OE3 and OE5 were subjected to NaCl (100 mM) stress for 24 h, and the whole seedlings were used for assaying the relative expression of the salt stress responsive genes. Actin was used as an internal control. Values ( n = 9) are mean ± SE and different letters on the histograms indicate that the values differ significantly (two-way ANOVA; P

Techniques Used: Expressing, RNA Extraction

12) Product Images from "Gut and blood differ in constitutive blocks to HIV transcription, suggesting tissue-specific differences in the mechanisms that govern HIV latency"

Article Title: Gut and blood differ in constitutive blocks to HIV transcription, suggesting tissue-specific differences in the mechanisms that govern HIV latency

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1007357

The HIV genome and the targets for transcription profiling assays. This schematic shows the genetic organization of proviral HIV DNA and the HIV ‘transcription profiling’ assays targeting specific RNA sequence regions that provide insight into blocks to transcription. Some proposed mechanisms underlying the blocks to transcription initiation, elongation, and splicing are detailed.
Figure Legend Snippet: The HIV genome and the targets for transcription profiling assays. This schematic shows the genetic organization of proviral HIV DNA and the HIV ‘transcription profiling’ assays targeting specific RNA sequence regions that provide insight into blocks to transcription. Some proposed mechanisms underlying the blocks to transcription initiation, elongation, and splicing are detailed.

Techniques Used: Sequencing

HIV RNA levels and HIV RNA/DNA ratios reveal blocks to elongation, distal transcription, and multiple-splicing in PBMCs and intact gut biopsies. Read-through, total (TAR), 5’ elongated (R-U5/pre-Gag; “Long LTR”), Nef, polyadenylated (PolyA), and multiply-spliced Tat-Rev (MS Tat-Rev) HIV RNAs were measured in (A) PBMCs; and (B) intact rectal biopsies ( n = 9 ART-suppressed individuals). 2C-D: Levels of HIV transcription per provirus are lower in the gut than the blood. Levels of each HIV RNA were normalized to HIV DNA from the same sample as measured by (C) ddPCR for the corresponding DNA sequence region (PolyA was normalized to the Read-through assay, which employs the same forward primer/probe), except for MS Tat-Rev where there is no DNA equivalent; or (D) ddPCR for the Long LTR assay, which is present once in each intact provirus. Bars indicate the median. Comparisons between transcripts were performed using the Wilcoxon signed-rank test.
Figure Legend Snippet: HIV RNA levels and HIV RNA/DNA ratios reveal blocks to elongation, distal transcription, and multiple-splicing in PBMCs and intact gut biopsies. Read-through, total (TAR), 5’ elongated (R-U5/pre-Gag; “Long LTR”), Nef, polyadenylated (PolyA), and multiply-spliced Tat-Rev (MS Tat-Rev) HIV RNAs were measured in (A) PBMCs; and (B) intact rectal biopsies ( n = 9 ART-suppressed individuals). 2C-D: Levels of HIV transcription per provirus are lower in the gut than the blood. Levels of each HIV RNA were normalized to HIV DNA from the same sample as measured by (C) ddPCR for the corresponding DNA sequence region (PolyA was normalized to the Read-through assay, which employs the same forward primer/probe), except for MS Tat-Rev where there is no DNA equivalent; or (D) ddPCR for the Long LTR assay, which is present once in each intact provirus. Bars indicate the median. Comparisons between transcripts were performed using the Wilcoxon signed-rank test.

Techniques Used: Mass Spectrometry, Sequencing

Stability of HIV transcripts ex vivo . Peripheral CD4+ T cells were isolated from an ART-suppressed individual and treated with the RNA polymerase II (RNA Pol II) inhibitors (A) Triptolide [100 nM] or (B) Actinomycin D [5 mg/mL] to arrest de novo cellular and viral transcription. HIV transcripts (Read-through, TAR, Long LTR, Nef, PolyA, and MS Tat-Rev) were quantified using RT-ddPCR from cells harvested at various time points post-treatment. Levels of each HIV RNA were expressed as a proportion of the level at time t = 0 (shown) and the half-lives were determined using a one-phase exponential decay model. Data normalized to DNA mass are shown.
Figure Legend Snippet: Stability of HIV transcripts ex vivo . Peripheral CD4+ T cells were isolated from an ART-suppressed individual and treated with the RNA polymerase II (RNA Pol II) inhibitors (A) Triptolide [100 nM] or (B) Actinomycin D [5 mg/mL] to arrest de novo cellular and viral transcription. HIV transcripts (Read-through, TAR, Long LTR, Nef, PolyA, and MS Tat-Rev) were quantified using RT-ddPCR from cells harvested at various time points post-treatment. Levels of each HIV RNA were expressed as a proportion of the level at time t = 0 (shown) and the half-lives were determined using a one-phase exponential decay model. Data normalized to DNA mass are shown.

Techniques Used: Ex Vivo, Isolation, Mass Spectrometry

Levels of HIV DNA, 2-LTR circles, and blocks to transcription differ between CD4+ T cells from the gut and blood. (A) Levels of HIV DNA (Long LTR region) and 2-LTR circles were quantified in CD4+ T cells from the blood and rectum using ddPCR and expressed as copies per million CD4+ T cells (normalized by TERT). (B) The average levels per provirus of each transcript (ratio of each HIV RNA to the Long LTR HIV DNA) were measured in CD4+ T cells isolated from the blood and rectum (n = 7 matched individuals) . (C) The ratio of TAR to Long LTR RNA was determined to compare the block to transcriptional elongation in CD4+ T cells from the blood and rectum. (D) The ratio of Read-through to Long LTR RNA was determined to assess the contribution of transcriptional interference to the block to HIV transcription initiation in rectal CD4+ T cells relative to peripheral CD4+ T cells. Bars represent medians. Comparisons between transcripts were performed using the Wilcoxon signed-rank test.
Figure Legend Snippet: Levels of HIV DNA, 2-LTR circles, and blocks to transcription differ between CD4+ T cells from the gut and blood. (A) Levels of HIV DNA (Long LTR region) and 2-LTR circles were quantified in CD4+ T cells from the blood and rectum using ddPCR and expressed as copies per million CD4+ T cells (normalized by TERT). (B) The average levels per provirus of each transcript (ratio of each HIV RNA to the Long LTR HIV DNA) were measured in CD4+ T cells isolated from the blood and rectum (n = 7 matched individuals) . (C) The ratio of TAR to Long LTR RNA was determined to compare the block to transcriptional elongation in CD4+ T cells from the blood and rectum. (D) The ratio of Read-through to Long LTR RNA was determined to assess the contribution of transcriptional interference to the block to HIV transcription initiation in rectal CD4+ T cells relative to peripheral CD4+ T cells. Bars represent medians. Comparisons between transcripts were performed using the Wilcoxon signed-rank test.

Techniques Used: Isolation, Blocking Assay

13) Product Images from "Integrating host response and unbiased microbe detection for lower respiratory tract infection diagnosis in critically ill adults"

Article Title: Integrating host response and unbiased microbe detection for lower respiratory tract infection diagnosis in critically ill adults

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

doi: 10.1073/pnas.1809700115

Workflow for distinguishing LRTI pathogens from commensal respiratory microbiota using an algorithmic approach. ( A ) Projection of microbial relative abundance in log reads per million reads sequenced (rpm) by RNA sequencing (RNA-seq) ( x axis) versus DNA sequencing (DNA-seq) ( y axis) for representative cases. In the LRTI +C+M group, pathogens identified by standard clinical microbiology (filled shapes) had higher overall relative abundance compared with other taxa detected by sequencing (open shapes). The largest score differential between ranked microbes (max Δrpm) was used as a threshold to identify high-scoring taxa, distinct from the other microbes based on abundance (line with arrows). Red indicates taxa represented in the reference list of established LRTI pathogens. ( B ) Receiver operating characteristic (ROC) curve demonstrating logistic regression model (LRM) performance for detecting pathogens versus commensal microbiota in both the derivation and validation cohorts. The gray ROC curve and shaded region indicate results from 1,000 rounds of training and testing on randomized sets the derivation cohort. The blue and green lines indicate predictions using leave-one-patient-out cross-validation (LOPO-CV) on the derivation and validation on the validation cohort, respectively. ( C ) Microbes predicted by the LRM to represent putative pathogens. The x axis represents combined RNA-seq and DNA-seq relative abundance, and the y axis indicates pathogen probability. The dashed line reflects the optimized probability threshold for pathogen assignment. Red filled circles: microbes predicted by LRM to represent putative LRTI pathogens that were also identified by conventional microbiologic tests. Blue filled circles: microbes predicted to represent putative LRTI pathogens by LRM only. Blue open circles: microbes identified by NGS but not predicted by the LRM to represent putative pathogens. Red open circles: microbes identified using NGS and by standard microbiologic testing but not predicted to be putative pathogens. Dark red outlined circles: microbes detected as part of a polymicrobial culture.
Figure Legend Snippet: Workflow for distinguishing LRTI pathogens from commensal respiratory microbiota using an algorithmic approach. ( A ) Projection of microbial relative abundance in log reads per million reads sequenced (rpm) by RNA sequencing (RNA-seq) ( x axis) versus DNA sequencing (DNA-seq) ( y axis) for representative cases. In the LRTI +C+M group, pathogens identified by standard clinical microbiology (filled shapes) had higher overall relative abundance compared with other taxa detected by sequencing (open shapes). The largest score differential between ranked microbes (max Δrpm) was used as a threshold to identify high-scoring taxa, distinct from the other microbes based on abundance (line with arrows). Red indicates taxa represented in the reference list of established LRTI pathogens. ( B ) Receiver operating characteristic (ROC) curve demonstrating logistic regression model (LRM) performance for detecting pathogens versus commensal microbiota in both the derivation and validation cohorts. The gray ROC curve and shaded region indicate results from 1,000 rounds of training and testing on randomized sets the derivation cohort. The blue and green lines indicate predictions using leave-one-patient-out cross-validation (LOPO-CV) on the derivation and validation on the validation cohort, respectively. ( C ) Microbes predicted by the LRM to represent putative pathogens. The x axis represents combined RNA-seq and DNA-seq relative abundance, and the y axis indicates pathogen probability. The dashed line reflects the optimized probability threshold for pathogen assignment. Red filled circles: microbes predicted by LRM to represent putative LRTI pathogens that were also identified by conventional microbiologic tests. Blue filled circles: microbes predicted to represent putative LRTI pathogens by LRM only. Blue open circles: microbes identified by NGS but not predicted by the LRM to represent putative pathogens. Red open circles: microbes identified using NGS and by standard microbiologic testing but not predicted to be putative pathogens. Dark red outlined circles: microbes detected as part of a polymicrobial culture.

Techniques Used: RNA Sequencing Assay, DNA Sequencing, Sequencing, Next-Generation Sequencing

Study overview and analysis workflow. Patients with acute respiratory failure were enrolled within 72 h of ICU admission, and TA samples were collected and underwent both RNA sequencing (RNA-seq) and shotgun DNA sequencing (DNA-seq). Post hoc clinical adjudication blinded to mNGS results identified patients with LRTI defined by clinical and microbiologic criteria (LRTI +C+M ); LRTI defined by clinical criteria only (LRTI +C ); patients with noninfectious reasons for acute respiratory failure (no-LRTI); and respiratory failure due to unknown cause (unk-LRTI). The LRTI +C+M and no-LRTI groups were divided into derivation and validation cohorts. To detect pathogens and differentiate them from a background of commensal microbiota, we developed two models: a rules-based model (RBM) and a logistic regression model (LRM). LRTI probability was next evaluated with ( i ) a pathogen metric, ( ii ) a lung microbiome diversity metric, and ( iii ) a 12-gene host transcriptional classifier. Models were then combined and optimized for LRTI rule out.
Figure Legend Snippet: Study overview and analysis workflow. Patients with acute respiratory failure were enrolled within 72 h of ICU admission, and TA samples were collected and underwent both RNA sequencing (RNA-seq) and shotgun DNA sequencing (DNA-seq). Post hoc clinical adjudication blinded to mNGS results identified patients with LRTI defined by clinical and microbiologic criteria (LRTI +C+M ); LRTI defined by clinical criteria only (LRTI +C ); patients with noninfectious reasons for acute respiratory failure (no-LRTI); and respiratory failure due to unknown cause (unk-LRTI). The LRTI +C+M and no-LRTI groups were divided into derivation and validation cohorts. To detect pathogens and differentiate them from a background of commensal microbiota, we developed two models: a rules-based model (RBM) and a logistic regression model (LRM). LRTI probability was next evaluated with ( i ) a pathogen metric, ( ii ) a lung microbiome diversity metric, and ( iii ) a 12-gene host transcriptional classifier. Models were then combined and optimized for LRTI rule out.

Techniques Used: RNA Sequencing Assay, DNA Sequencing

14) Product Images from "Differentiation of Human Dental Stem Cells Reveal a Role for microRNA-218"

Article Title: Differentiation of Human Dental Stem Cells Reveal a Role for microRNA-218

Journal: Journal of periodontal research

doi: 10.1111/jre.12086

qRT-PCR results of OCT4 and NANOG expression-OCT4 and NANOG levels in DPSC, PDLSC, and GINGSC populations are shown relative to BMSC levels. RNA was harvested from isolated dental stem cells or after 21 days in osteogenesis differentiation medium (DF, Differentiated cells). All data was normalized to GAPDH to account for RNA concentration differences in the samples.
Figure Legend Snippet: qRT-PCR results of OCT4 and NANOG expression-OCT4 and NANOG levels in DPSC, PDLSC, and GINGSC populations are shown relative to BMSC levels. RNA was harvested from isolated dental stem cells or after 21 days in osteogenesis differentiation medium (DF, Differentiated cells). All data was normalized to GAPDH to account for RNA concentration differences in the samples.

Techniques Used: Quantitative RT-PCR, Expressing, Isolation, Concentration Assay

15) Product Images from "Transcriptional and Mutational Analyses of the Streptomyces lividans recX Gene and Its Interference with RecA Activity"

Article Title: Transcriptional and Mutational Analyses of the Streptomyces lividans recX Gene and Its Interference with RecA Activity

Journal: Journal of Bacteriology

doi:

Transcriptional analysis of the recA-recX operon by RT-PCR. After induction with 25 μg of MMS ml −1 , RNA was isolated from S. lividans TK64 at different intervals and used for RT-PCR with different primer combinations. (A) recA -specific primers. (B) recA-recX cotranscript-specific primers. (C) recX -specific primers. (D) Control reaction using glnA -specific primers. (E) Control reaction without RT. Lanes: DNA, control PCR with genomic DNA as template; 0, without induction; 20, 40, and 60, 20, 40, and 60 min after induction, respectively; M, size standard (1-kb ladder [Boehringer]: 12,216, 11,198, 10,180, 9,162, 8,144, 7,126, 6,108, 5,090, 4,072, 3,054, 2,036, 1,636, 1,018, 517, 506, 396, 344, 298, 220, 201, 154, 134, and 75 bp).
Figure Legend Snippet: Transcriptional analysis of the recA-recX operon by RT-PCR. After induction with 25 μg of MMS ml −1 , RNA was isolated from S. lividans TK64 at different intervals and used for RT-PCR with different primer combinations. (A) recA -specific primers. (B) recA-recX cotranscript-specific primers. (C) recX -specific primers. (D) Control reaction using glnA -specific primers. (E) Control reaction without RT. Lanes: DNA, control PCR with genomic DNA as template; 0, without induction; 20, 40, and 60, 20, 40, and 60 min after induction, respectively; M, size standard (1-kb ladder [Boehringer]: 12,216, 11,198, 10,180, 9,162, 8,144, 7,126, 6,108, 5,090, 4,072, 3,054, 2,036, 1,636, 1,018, 517, 506, 396, 344, 298, 220, 201, 154, 134, and 75 bp).

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

16) Product Images from "CTLA-4+PD-1− memory CD4+ T cells critically contribute to viral persistence in antiretroviral therapy-suppressed, SIV-infected rhesus macaques"

Article Title: CTLA-4+PD-1− memory CD4+ T cells critically contribute to viral persistence in antiretroviral therapy-suppressed, SIV-infected rhesus macaques

Journal: Immunity

doi: 10.1016/j.immuni.2017.09.018

CTLA-4 + PD-1 − memory CD4 + T cells harbor higher amounts of SIV DNA following ART-mediated, viral load suppression ( A )Study design. Ten RMs were infected i.v. with 1000 TCID50 SIVmac251 (day 0), and at 7 weeks post-infection, initiated ART (PMPA, FTC, raltegravir, and ritonavir-boosted darunavir). All animals were maintained on ART regimen until plasma viremia was undetectable for at least 3 months. Peripheral blood (WB), rectal biopsy (Gut), and lymph node (LN) biopsies were collected at the indicated time points and multiple organs were harvested at elective necropsy. Sorting of memory CD4 + T cells by Co-IR expression was performed at two time points during ART: first, at Mid ART (approximately 1 month following undetectable viremia); and second, at necropsy. ( B ) Plasma viral loads are shown for the 10 individual RMs, quantified using the standard qRT-PCR assay (limit of detection, LOD, of 60 SIV RNA copies/mL of plasma represented by the horizontal dotted line). Undetectable measurements are plotted as one-half of the LOD (30 copies/mL). ( C ) Frequencies (of live CD3 + T cells) of CD4 + T cells were longitudinally measured in WB, LN, and gut biopsies. The gray shaded area represents time on ART; Nx represents the measured values from animal necropsy. Repeated-measures analyses were performed using a means model (SAS Mixed Procedure, version 9.4) to determine statistical significance, with indicated tests of significance representing comparison to pre-SIV infection (WB, Gut) or pre-ART initiation (LN). ( D ) Representative SIV DNA quantities in the PBMCs, LN, spleen, and gut tissues for an individual RM (RLr10) after 206 days of viral load suppression (n=9). ( E ) Cell-associated SIV GAG .
Figure Legend Snippet: CTLA-4 + PD-1 − memory CD4 + T cells harbor higher amounts of SIV DNA following ART-mediated, viral load suppression ( A )Study design. Ten RMs were infected i.v. with 1000 TCID50 SIVmac251 (day 0), and at 7 weeks post-infection, initiated ART (PMPA, FTC, raltegravir, and ritonavir-boosted darunavir). All animals were maintained on ART regimen until plasma viremia was undetectable for at least 3 months. Peripheral blood (WB), rectal biopsy (Gut), and lymph node (LN) biopsies were collected at the indicated time points and multiple organs were harvested at elective necropsy. Sorting of memory CD4 + T cells by Co-IR expression was performed at two time points during ART: first, at Mid ART (approximately 1 month following undetectable viremia); and second, at necropsy. ( B ) Plasma viral loads are shown for the 10 individual RMs, quantified using the standard qRT-PCR assay (limit of detection, LOD, of 60 SIV RNA copies/mL of plasma represented by the horizontal dotted line). Undetectable measurements are plotted as one-half of the LOD (30 copies/mL). ( C ) Frequencies (of live CD3 + T cells) of CD4 + T cells were longitudinally measured in WB, LN, and gut biopsies. The gray shaded area represents time on ART; Nx represents the measured values from animal necropsy. Repeated-measures analyses were performed using a means model (SAS Mixed Procedure, version 9.4) to determine statistical significance, with indicated tests of significance representing comparison to pre-SIV infection (WB, Gut) or pre-ART initiation (LN). ( D ) Representative SIV DNA quantities in the PBMCs, LN, spleen, and gut tissues for an individual RM (RLr10) after 206 days of viral load suppression (n=9). ( E ) Cell-associated SIV GAG .

Techniques Used: Infection, Western Blot, Expressing, Quantitative RT-PCR

17) Product Images from "Increased T cell infiltration elicited by Erk5deletion in a Pten-deficient mouse model of prostate carcinogenesis"

Article Title: Increased T cell infiltration elicited by Erk5deletion in a Pten-deficient mouse model of prostate carcinogenesis

Journal: Cancer research

doi: 10.1158/0008-5472.CAN-16-2565

Histopathological analysis revealed that Pten fl/fl Erk5 fl/fl prostate tumours were less proliferative compared to Pten fl/fl prostate tumours and that Pten fl/fl prostate tumours expressed more Erk5 compared to WT prostate ( A ) H+E staining was performed to examine the tissue histology and Ki67 IHC was employed to investigate the level of cellular proliferation in prostates from WT, Erk5 fl/fl , Pten fl/fl and Pten fl/fl Erk5 fl/fl mice (n=minimum of 3 for each genotype). Representative micrographs of H+E and Ki67 staining for each genotype are shown (main images 10x; insets are higher magnification (40x) view of prostate epithelium). ( B ) % Ki67 positive cells in each sample were scored using an automated method (Leica software). 12 representative fields of view per sample were scored for n=6 samples for each genotype at end point. (WT all 15 months; Erk5 fl/fl all 15 months; Pten fl/fl age range 11-15 months; Pten fl/fl Erk5 fl/fl all 15 months) ( C ) QPCR analysis of RNA isolated from mouse prostate epithelium by LCM in WT, Erk5 fl/fl , Pten fl/fl and Pten fl/fl Erk5 fl/fl mice (n=3 for each genotype) for Erk5 Exon 4 (the floxed allele) and Pten . ( D ) RNA ISH analysis by RNA Scope was utilised to examine the level of Erk5 expression in WT prostate and Pten fl/fl prostate tumours (n=3 for each genotype). Representative micrographs (100x) of RNA Scope staining for Erk5 , Ppib (positive control for RNA integrity) and Dapb (negative control for probe specificity) of WT prostate and Pten fl/fl tumours at end point are presented. Shown in ( B ) and ( C ) are the means; error bars represent SEM; t test (unpaired, 2 tailed) was used to calculate p values; key significant changes (p
Figure Legend Snippet: Histopathological analysis revealed that Pten fl/fl Erk5 fl/fl prostate tumours were less proliferative compared to Pten fl/fl prostate tumours and that Pten fl/fl prostate tumours expressed more Erk5 compared to WT prostate ( A ) H+E staining was performed to examine the tissue histology and Ki67 IHC was employed to investigate the level of cellular proliferation in prostates from WT, Erk5 fl/fl , Pten fl/fl and Pten fl/fl Erk5 fl/fl mice (n=minimum of 3 for each genotype). Representative micrographs of H+E and Ki67 staining for each genotype are shown (main images 10x; insets are higher magnification (40x) view of prostate epithelium). ( B ) % Ki67 positive cells in each sample were scored using an automated method (Leica software). 12 representative fields of view per sample were scored for n=6 samples for each genotype at end point. (WT all 15 months; Erk5 fl/fl all 15 months; Pten fl/fl age range 11-15 months; Pten fl/fl Erk5 fl/fl all 15 months) ( C ) QPCR analysis of RNA isolated from mouse prostate epithelium by LCM in WT, Erk5 fl/fl , Pten fl/fl and Pten fl/fl Erk5 fl/fl mice (n=3 for each genotype) for Erk5 Exon 4 (the floxed allele) and Pten . ( D ) RNA ISH analysis by RNA Scope was utilised to examine the level of Erk5 expression in WT prostate and Pten fl/fl prostate tumours (n=3 for each genotype). Representative micrographs (100x) of RNA Scope staining for Erk5 , Ppib (positive control for RNA integrity) and Dapb (negative control for probe specificity) of WT prostate and Pten fl/fl tumours at end point are presented. Shown in ( B ) and ( C ) are the means; error bars represent SEM; t test (unpaired, 2 tailed) was used to calculate p values; key significant changes (p

Techniques Used: Staining, Immunohistochemistry, Mouse Assay, Software, Real-time Polymerase Chain Reaction, Isolation, Laser Capture Microdissection, In Situ Hybridization, Expressing, Positive Control, Negative Control

18) Product Images from "Hedgehog signaling has a protective effect in glucocorticoid-induced mouse neonatal brain injury through an 11?HSD2-dependent mechanism"

Article Title: Hedgehog signaling has a protective effect in glucocorticoid-induced mouse neonatal brain injury through an 11?HSD2-dependent mechanism

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI36376

Shh signaling upregulates transcription of 11 β HSD2 in vitro and in vivo. ( A ) Whole RNA was isolated from WT and Math1cre,SmoM2 CGNP cultures treated with vehicle, Shh, or Shh plus 40 μM Dex for 24 h. Quantitative PCR analysis showed that Shh treatment potently induced the Shh targets N-myc and Gli1 as well as 11 β HSD2 expression in WT CGNP cultures. Math1cre,SmoM2 CGNPs showed increased expression of N-myc , Gli1 , and 11 β HSD2 in both vehicle and Shh groups and was unchanged after Dex treatment. 11 β HSD1 expression was below detectable levels. ( B ) In vivo, N-myc , Gli1 , and 11 β HSD2 levels were upregulated in the Math1cre,SmoM2 cerebellum (CB). ( C ) In situ hybridization showing specific expression of 11 β HSD2 in the EGL of the P7 WT and Math1cre,SmoM2 cerebellum. Original magnification, ×400.
Figure Legend Snippet: Shh signaling upregulates transcription of 11 β HSD2 in vitro and in vivo. ( A ) Whole RNA was isolated from WT and Math1cre,SmoM2 CGNP cultures treated with vehicle, Shh, or Shh plus 40 μM Dex for 24 h. Quantitative PCR analysis showed that Shh treatment potently induced the Shh targets N-myc and Gli1 as well as 11 β HSD2 expression in WT CGNP cultures. Math1cre,SmoM2 CGNPs showed increased expression of N-myc , Gli1 , and 11 β HSD2 in both vehicle and Shh groups and was unchanged after Dex treatment. 11 β HSD1 expression was below detectable levels. ( B ) In vivo, N-myc , Gli1 , and 11 β HSD2 levels were upregulated in the Math1cre,SmoM2 cerebellum (CB). ( C ) In situ hybridization showing specific expression of 11 β HSD2 in the EGL of the P7 WT and Math1cre,SmoM2 cerebellum. Original magnification, ×400.

Techniques Used: In Vitro, In Vivo, Isolation, Real-time Polymerase Chain Reaction, Expressing, In Situ Hybridization

19) Product Images from "Hairpin RNA induces secondary small interfering RNA synthesis and silencing in trans in fission yeast"

Article Title: Hairpin RNA induces secondary small interfering RNA synthesis and silencing in trans in fission yeast

Journal: EMBO Reports

doi: 10.1038/embor.2009.273

GFP-HP induces trans -silencing of target genes. ( A ) Growth assay demonstrating that the ura4 + –GFP fusion gene is silenced by expression of GFP-HP in wild-type but not the indicated mutant strains. Silencing is indicated by growth on counter-selective 5-FOA. ( B ) Colony colour assay to monitor silencing of ade6 + –GFP by the GFP-HP on indicator plates. Expression of GFP-HP induces unstable silencing of the ade6 + –GFP reporter gene. Silencing is indicated by the appearance of red colonies; 19% of colonies were red. ( C ) qRT–PCR of ura4 + –GFP relative to act1 + indicates that ura4 + –GFP levels are reduced in cells expressing GFP-HP when grown in the presence of counter-selective 5-FOA, which selects for cells with silent ura4 + . 5-FOA, 5-fluorootic acid; GFP, green fluorescent protein; GFP-HP, GFP hairpin; qRT–PCR, quantitative reverse transcriptase PCR; siRNA, small interfering RNA; wt, wild type.
Figure Legend Snippet: GFP-HP induces trans -silencing of target genes. ( A ) Growth assay demonstrating that the ura4 + –GFP fusion gene is silenced by expression of GFP-HP in wild-type but not the indicated mutant strains. Silencing is indicated by growth on counter-selective 5-FOA. ( B ) Colony colour assay to monitor silencing of ade6 + –GFP by the GFP-HP on indicator plates. Expression of GFP-HP induces unstable silencing of the ade6 + –GFP reporter gene. Silencing is indicated by the appearance of red colonies; 19% of colonies were red. ( C ) qRT–PCR of ura4 + –GFP relative to act1 + indicates that ura4 + –GFP levels are reduced in cells expressing GFP-HP when grown in the presence of counter-selective 5-FOA, which selects for cells with silent ura4 + . 5-FOA, 5-fluorootic acid; GFP, green fluorescent protein; GFP-HP, GFP hairpin; qRT–PCR, quantitative reverse transcriptase PCR; siRNA, small interfering RNA; wt, wild type.

Techniques Used: Growth Assay, Expressing, Mutagenesis, Quantitative RT-PCR, Polymerase Chain Reaction, Small Interfering RNA

GFP-HP induces chromatin modifications. ( A ) H3K9me2 and Swi6 ChIP analyses. The enrichment of ura4 + –GFP and cen-otr compared with act1 + was calculated by quantitative PCR and subsequently the ratio of enrichment in cells expressing GFP-HP relative to cells without the hairpin was plotted. The position of the ura4 + -specific primers (arrowheads) for ura4 + –GFP is shown. ( B ) H3K9me2 ChIP analyses at the GFP-HP locus. Quantitative PCR allowed the quantification of H3K9me2 enrichment on ura4 + – GFP , GFP-HP and cen-otr relative to act1 + . The diagram shows the position of the GFP-HP-specific primers used (arrowheads). ura4–GFP primers were as in ( A ). ( C ) Strand-specific RT–PCR assay for transcripts from ura4 + –GFP . Diagram indicating the primer pairs used in RT–PCR to detect transcripts across the middle (mid) and 3′ end (end) of the ura4 + –GFP fusion gene. Primers used for cDNA synthesis of antisense (upper arrowheads) or sense (lower arrowheads) transcripts. RT–PCR was carried out with an equal amount of total RNAs from strains with and without the GFP-HP. RT–PCR with act1 + primers was performed as a control. as, antisense; cDNA, complementary DNA; ChIP, chromatin immunoprecipitation; GFP, green fluorescent protein; GFP-HP, GFP hairpin; RT–PCR, reverse transcriptase PCR; s, sense; siRNA, small interfering RNA.
Figure Legend Snippet: GFP-HP induces chromatin modifications. ( A ) H3K9me2 and Swi6 ChIP analyses. The enrichment of ura4 + –GFP and cen-otr compared with act1 + was calculated by quantitative PCR and subsequently the ratio of enrichment in cells expressing GFP-HP relative to cells without the hairpin was plotted. The position of the ura4 + -specific primers (arrowheads) for ura4 + –GFP is shown. ( B ) H3K9me2 ChIP analyses at the GFP-HP locus. Quantitative PCR allowed the quantification of H3K9me2 enrichment on ura4 + – GFP , GFP-HP and cen-otr relative to act1 + . The diagram shows the position of the GFP-HP-specific primers used (arrowheads). ura4–GFP primers were as in ( A ). ( C ) Strand-specific RT–PCR assay for transcripts from ura4 + –GFP . Diagram indicating the primer pairs used in RT–PCR to detect transcripts across the middle (mid) and 3′ end (end) of the ura4 + –GFP fusion gene. Primers used for cDNA synthesis of antisense (upper arrowheads) or sense (lower arrowheads) transcripts. RT–PCR was carried out with an equal amount of total RNAs from strains with and without the GFP-HP. RT–PCR with act1 + primers was performed as a control. as, antisense; cDNA, complementary DNA; ChIP, chromatin immunoprecipitation; GFP, green fluorescent protein; GFP-HP, GFP hairpin; RT–PCR, reverse transcriptase PCR; s, sense; siRNA, small interfering RNA.

Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Small Interfering RNA

20) Product Images from "Utility of siRNA against Keap1 as a strategy to stimulate a cancer chemopreventive phenotype"

Article Title: Utility of siRNA against Keap1 as a strategy to stimulate a cancer chemopreventive phenotype

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

doi: 10.1073/pnas.0501475102

Keap1 siRNA causes induction of antioxidant and detoxication proteins. HaCaT cells were transfected with either Lipofectamine alone (Mock), 100 nM Target1, or 100 nM scrambled (Scrm) siRNA and then harvested 48 h later. In addition, nontransfected HaCaT cells were treated with 0.01% DMSO or 5 μM Sul (in DMSO to a final concentration of 0.01%) before being harvested 24 h later. ( A ) Western blotting was performed on duplicate 10-μg protein portions of cell lysates. ( B - D ) TaqMan chemistry was performed on total RNA to quantify message levels for AKR1C1/2 ( B ), GCLC ( C ), and NQO1 ( D ); the data are presented as relative transcriptional units (RTu), and each is the mean of duplicate experiments ± SE.
Figure Legend Snippet: Keap1 siRNA causes induction of antioxidant and detoxication proteins. HaCaT cells were transfected with either Lipofectamine alone (Mock), 100 nM Target1, or 100 nM scrambled (Scrm) siRNA and then harvested 48 h later. In addition, nontransfected HaCaT cells were treated with 0.01% DMSO or 5 μM Sul (in DMSO to a final concentration of 0.01%) before being harvested 24 h later. ( A ) Western blotting was performed on duplicate 10-μg protein portions of cell lysates. ( B - D ) TaqMan chemistry was performed on total RNA to quantify message levels for AKR1C1/2 ( B ), GCLC ( C ), and NQO1 ( D ); the data are presented as relative transcriptional units (RTu), and each is the mean of duplicate experiments ± SE.

Techniques Used: Transfection, Concentration Assay, Western Blot

Knockdown of Keap1 with siRNA. HaCaT cells were transfected with Keap1 siRNA and scrambled siRNA. Levels of Keap1 mRNA were quantified by RT-PCR TaqMan using 18S rRNA as an internal standard. Each experiment was performed in triplicate, and data are presented as the mean relative transcriptional units (RTu) ± SE. ( A ) HaCaT cells were transfected with 100 nM concentrations each of Target1, Target2, or scrambled siRNA 48 h before RNA was isolated and analyzed. ( B ) Different concentrations (Conc) of Target1 were used to transfect HaCaT cells 48 h before RNA was isolated and analyzed. ( C ) Cells were transfected with 100 nM Target1, and RNA was analyzed 24, 48, and 72 h after transfection.
Figure Legend Snippet: Knockdown of Keap1 with siRNA. HaCaT cells were transfected with Keap1 siRNA and scrambled siRNA. Levels of Keap1 mRNA were quantified by RT-PCR TaqMan using 18S rRNA as an internal standard. Each experiment was performed in triplicate, and data are presented as the mean relative transcriptional units (RTu) ± SE. ( A ) HaCaT cells were transfected with 100 nM concentrations each of Target1, Target2, or scrambled siRNA 48 h before RNA was isolated and analyzed. ( B ) Different concentrations (Conc) of Target1 were used to transfect HaCaT cells 48 h before RNA was isolated and analyzed. ( C ) Cells were transfected with 100 nM Target1, and RNA was analyzed 24, 48, and 72 h after transfection.

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

21) Product Images from "Comprehensive analysis of microRNA signature of mouse pancreatic acini: overexpression of miR-21-3p in acute pancreatitis"

Article Title: Comprehensive analysis of microRNA signature of mouse pancreatic acini: overexpression of miR-21-3p in acute pancreatitis

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

doi: 10.1152/ajpgi.00191.2016

Expression of miR-21-3p in human pancreatic acinar cells. Human acinar cells were treated in vitro with 500 μM TLC-S or 1 mM carbachol (CCH) for 1 h, total RNA was isolated, and qPCR was done using miRNA-specific primer and SYBR Green dye ( n = 3) (* P
Figure Legend Snippet: Expression of miR-21-3p in human pancreatic acinar cells. Human acinar cells were treated in vitro with 500 μM TLC-S or 1 mM carbachol (CCH) for 1 h, total RNA was isolated, and qPCR was done using miRNA-specific primer and SYBR Green dye ( n = 3) (* P

Techniques Used: Expressing, In Vitro, Thin Layer Chromatography, Isolation, Real-time Polymerase Chain Reaction, SYBR Green Assay

22) Product Images from "Bex1 is involved in the regeneration of axons after injury"

Article Title: Bex1 is involved in the regeneration of axons after injury

Journal: Journal of Neurochemistry

doi: 10.1111/j.1471-4159.2010.06960.x

Bex1 upregulation in MNs after axonal injury a) MNs from the spinal cord of MPZ-KO and MAG-KO mice and mice subjected to SN-crush injury or demyelination were dissected by Laser capture microdissection and their total RNA was used for quantitative real-time PCR. n =3, error bars represent SEM, * p
Figure Legend Snippet: Bex1 upregulation in MNs after axonal injury a) MNs from the spinal cord of MPZ-KO and MAG-KO mice and mice subjected to SN-crush injury or demyelination were dissected by Laser capture microdissection and their total RNA was used for quantitative real-time PCR. n =3, error bars represent SEM, * p

Techniques Used: Mouse Assay, Laser Capture Microdissection, Real-time Polymerase Chain Reaction

23) Product Images from "Cartilage Oligomeric Matrix Protein-Deficient Mice Have Normal Skeletal Development"

Article Title: Cartilage Oligomeric Matrix Protein-Deficient Mice Have Normal Skeletal Development

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.22.12.4366-4371.2002

Northern blot and RT-PCR. (A) Northern blot analysis of RNA isolated from the processus xiphoideus of wild-type (wt) and mutant (m) littermates. Filters were hybridized with TSP-1, TSP-3, TSP-4, and a COMP probe. A glyceraldehyde phosphodehydrogenase (GAPDH) probe was used as a control. (B) RT-PCR analysis of COMP mRNA. COMP cDNA was reverse transcribed from processus xiphoideus poly(A) + RNA by using reverse transcriptase. PCR primers located upstream of the Hin dIII site used for insertion of the pGKNeo gene were used in the amplification of wild-type (lane 1) and COMP-null (lane 2) cDNAs. As a control, cDNAs were amplified with wild-type (lane 3) and COMP-null (lane 4) fibromodulin primers. Lane 5, control amplification without prior reverse transcription of poly(A) + RNA from COMP-null mice with fibromodulin primers.
Figure Legend Snippet: Northern blot and RT-PCR. (A) Northern blot analysis of RNA isolated from the processus xiphoideus of wild-type (wt) and mutant (m) littermates. Filters were hybridized with TSP-1, TSP-3, TSP-4, and a COMP probe. A glyceraldehyde phosphodehydrogenase (GAPDH) probe was used as a control. (B) RT-PCR analysis of COMP mRNA. COMP cDNA was reverse transcribed from processus xiphoideus poly(A) + RNA by using reverse transcriptase. PCR primers located upstream of the Hin dIII site used for insertion of the pGKNeo gene were used in the amplification of wild-type (lane 1) and COMP-null (lane 2) cDNAs. As a control, cDNAs were amplified with wild-type (lane 3) and COMP-null (lane 4) fibromodulin primers. Lane 5, control amplification without prior reverse transcription of poly(A) + RNA from COMP-null mice with fibromodulin primers.

Techniques Used: Northern Blot, Reverse Transcription Polymerase Chain Reaction, Isolation, Mutagenesis, Polymerase Chain Reaction, Amplification, Mouse Assay

24) Product Images from "Mlc Is a Transcriptional Activator with a Key Role in Integrating Cyclic AMP Receptor Protein and Integration Host Factor Regulation of Leukotoxin RNA Synthesis in Aggregatibacter actinomycetemcomitans"

Article Title: Mlc Is a Transcriptional Activator with a Key Role in Integrating Cyclic AMP Receptor Protein and Integration Host Factor Regulation of Leukotoxin RNA Synthesis in Aggregatibacter actinomycetemcomitans

Journal: Journal of Bacteriology

doi: 10.1128/JB.02144-12

Chromosomal deletions define the region between −69 and −35 as an important control region in the leukotoxin promoter. Plasmids with the deletions indicated in the region upstream of the leukotoxin operon (diagrammed at the top of the figure) were transformed into A. actinomycetemcomitans ). For data in the graph shown, each sample measured was grown anaerobically to log phase, RNA was isolated, and leukotoxin RNA levels were measure by quantitative RT-PCR. The amount of ltxA mRNA in each sample was normalized to the level of pdxY mRNA in the same sample, and then the ltxA mRNA level in each mutant was compared to that of the parental JP2 cells.
Figure Legend Snippet: Chromosomal deletions define the region between −69 and −35 as an important control region in the leukotoxin promoter. Plasmids with the deletions indicated in the region upstream of the leukotoxin operon (diagrammed at the top of the figure) were transformed into A. actinomycetemcomitans ). For data in the graph shown, each sample measured was grown anaerobically to log phase, RNA was isolated, and leukotoxin RNA levels were measure by quantitative RT-PCR. The amount of ltxA mRNA in each sample was normalized to the level of pdxY mRNA in the same sample, and then the ltxA mRNA level in each mutant was compared to that of the parental JP2 cells.

Techniques Used: Transformation Assay, Isolation, Quantitative RT-PCR, Mutagenesis

25) Product Images from "Mlc Is a Transcriptional Activator with a Key Role in Integrating Cyclic AMP Receptor Protein and Integration Host Factor Regulation of Leukotoxin RNA Synthesis in Aggregatibacter actinomycetemcomitans"

Article Title: Mlc Is a Transcriptional Activator with a Key Role in Integrating Cyclic AMP Receptor Protein and Integration Host Factor Regulation of Leukotoxin RNA Synthesis in Aggregatibacter actinomycetemcomitans

Journal: Journal of Bacteriology

doi: 10.1128/JB.02144-12

An mlc mutant makes less leukotoxin protein and RNA. Samples were taken at log phase from aerobically (+) and anaerobically (−) grown cultures of the parent strain (JP2) and Δ mlc (AAM155) cells as well as the deletion strain with a plasmid carrying mlc + (the Δ mlc / mlc + strain). Cells were harvested by centrifugation, resuspended in SDS-PAGE loading buffer, boiled, and centrifuged, and the soluble material was electrophoresed on duplicate gels. (A) Gel stained with Coomassie brilliant blue. (B) Western blot analysis of total cell protein from the indicated strains using rabbit antileukotoxin antibody as the primary antibody. The arrows mark the position of leukotoxin. The numbers show the positions of molecular size markers (not shown) in kDa. (C) For each sample, total cell RNA was prepared from anaerobically grown log phase cells, and cDNA was synthesized with random hexamer primers and then used in quantitative RT-PCR. The level of ltxA mRNA in each sample is normalized to the level of pdxY mRNA in the same sample. The data are the average of the expression ratios from three biological replicates. The error bars are standard error, and the difference between the two samples is statistically significant ( P ≤ 0.05, by Student's t test).
Figure Legend Snippet: An mlc mutant makes less leukotoxin protein and RNA. Samples were taken at log phase from aerobically (+) and anaerobically (−) grown cultures of the parent strain (JP2) and Δ mlc (AAM155) cells as well as the deletion strain with a plasmid carrying mlc + (the Δ mlc / mlc + strain). Cells were harvested by centrifugation, resuspended in SDS-PAGE loading buffer, boiled, and centrifuged, and the soluble material was electrophoresed on duplicate gels. (A) Gel stained with Coomassie brilliant blue. (B) Western blot analysis of total cell protein from the indicated strains using rabbit antileukotoxin antibody as the primary antibody. The arrows mark the position of leukotoxin. The numbers show the positions of molecular size markers (not shown) in kDa. (C) For each sample, total cell RNA was prepared from anaerobically grown log phase cells, and cDNA was synthesized with random hexamer primers and then used in quantitative RT-PCR. The level of ltxA mRNA in each sample is normalized to the level of pdxY mRNA in the same sample. The data are the average of the expression ratios from three biological replicates. The error bars are standard error, and the difference between the two samples is statistically significant ( P ≤ 0.05, by Student's t test).

Techniques Used: Mutagenesis, Plasmid Preparation, Centrifugation, SDS Page, Staining, Western Blot, Synthesized, Random Hexamer Labeling, Quantitative RT-PCR, Expressing

The genetic interactions of IHF, CRP, and Mlc in altering leukotoxin transcription. After each sample was grown to log phase, total cell RNA was isolated, and cDNA was prepared with random hexamer primers and then used in quantitative RT-PCR. The level of ltxA mRNA in each sample is normalized to the level of pdxY mRNA in the same sample. The data are the average of the expression ratios from three biological replicates. The error bars are standard error. A single asterisk indicates samples for which the ratio of ltxA mRNA to pdxY mRNA is statistically significantly ( P
Figure Legend Snippet: The genetic interactions of IHF, CRP, and Mlc in altering leukotoxin transcription. After each sample was grown to log phase, total cell RNA was isolated, and cDNA was prepared with random hexamer primers and then used in quantitative RT-PCR. The level of ltxA mRNA in each sample is normalized to the level of pdxY mRNA in the same sample. The data are the average of the expression ratios from three biological replicates. The error bars are standard error. A single asterisk indicates samples for which the ratio of ltxA mRNA to pdxY mRNA is statistically significantly ( P

Techniques Used: Immunohistofluorescence, Isolation, Random Hexamer Labeling, Quantitative RT-PCR, Expressing

Quantitative RT-PCR of BamHI mutations define key regulatory elements within the leukotoxin promoter. Each of the indicated strains was grown anaerobically (A) or aerobically (B) to log phase, total RNA was isolated, and cDNA was prepared with random hexamer primers and then used in quantitative RT-PCR with ltxA - and pdxY -specific primers. The levels of ltxA mRNA in a single sample were normalized to pdxY (internal control) levels in the same sample. The value for a given strain is the average of the ratios of the ltxA mRNA to pdxY mRNA levels from three biological replicates (samples) of that strain. The error bars are the sample standard deviation. A single asterisk indicates a sample for which the ratio of ltxA mRNA to pdxY mRNA is statistically significantly ( P
Figure Legend Snippet: Quantitative RT-PCR of BamHI mutations define key regulatory elements within the leukotoxin promoter. Each of the indicated strains was grown anaerobically (A) or aerobically (B) to log phase, total RNA was isolated, and cDNA was prepared with random hexamer primers and then used in quantitative RT-PCR with ltxA - and pdxY -specific primers. The levels of ltxA mRNA in a single sample were normalized to pdxY (internal control) levels in the same sample. The value for a given strain is the average of the ratios of the ltxA mRNA to pdxY mRNA levels from three biological replicates (samples) of that strain. The error bars are the sample standard deviation. A single asterisk indicates a sample for which the ratio of ltxA mRNA to pdxY mRNA is statistically significantly ( P

Techniques Used: Quantitative RT-PCR, Isolation, Random Hexamer Labeling, Standard Deviation

26) Product Images from "Expression of Procyclin mRNAs during Cyclical Transmission of Trypanosoma brucei"

Article Title: Expression of Procyclin mRNAs during Cyclical Transmission of Trypanosoma brucei

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.0010022

Relative Amount of Procyclin mRNA in Different Life Cycle Stages RTQ-PCR was performed on RNA samples from bloodstream forms and midgut and salivary gland trypanosomes at various days post infection. β-tubulin was used as an internal control. The ratio of procyclin/tubulin mRNAs in a reference culture of procyclic forms was set at 1. Standard deviations derive from two to five replicas. RNA derived from independent infections (days 11–24) was quantitated separately.
Figure Legend Snippet: Relative Amount of Procyclin mRNA in Different Life Cycle Stages RTQ-PCR was performed on RNA samples from bloodstream forms and midgut and salivary gland trypanosomes at various days post infection. β-tubulin was used as an internal control. The ratio of procyclin/tubulin mRNAs in a reference culture of procyclic forms was set at 1. Standard deviations derive from two to five replicas. RNA derived from independent infections (days 11–24) was quantitated separately.

Techniques Used: Polymerase Chain Reaction, Infection, Derivative Assay

27) Product Images from "MiR-184 regulates insulin secretion through repression of Slc25a22"

Article Title: MiR-184 regulates insulin secretion through repression of Slc25a22

Journal: PeerJ

doi: 10.7717/peerj.162

SiRNAs for Slc25a22 inhibits glucose-induced insulin secretion in the MIN6 islet β-cell line. SiRNA for Slc25a22 (Slc25a22_2, Slc25a22_4) or control siRNA was transfected into the MIN6 islet β-cell line. (A) Expression of Slc25a22 mRNA. Quantitative RT-PCR analyses of the expression levels of Slc25a22 mRNA were performed 48 h after transfection of MIN6 cells with Slc25a22 (Slc25a22_2, Slc25a22_4) or control siRNA. The 18S ribosomal RNA was used to normalize the expression levels. Data show the mean + SD for n = 3 repeats. ** p
Figure Legend Snippet: SiRNAs for Slc25a22 inhibits glucose-induced insulin secretion in the MIN6 islet β-cell line. SiRNA for Slc25a22 (Slc25a22_2, Slc25a22_4) or control siRNA was transfected into the MIN6 islet β-cell line. (A) Expression of Slc25a22 mRNA. Quantitative RT-PCR analyses of the expression levels of Slc25a22 mRNA were performed 48 h after transfection of MIN6 cells with Slc25a22 (Slc25a22_2, Slc25a22_4) or control siRNA. The 18S ribosomal RNA was used to normalize the expression levels. Data show the mean + SD for n = 3 repeats. ** p

Techniques Used: Transfection, Expressing, Quantitative RT-PCR

The effect of MiR-184 on endogenous Slc25a22. (A) Expression of Slc25a22 mRNA. Quantitative RT-PCR analyses of the expression levels of Slc25a22 mRNA were performed 48 h after transfection of MIN6 cells with miR-184 or control miRNA. The 18S ribosomal RNA was used to normalize the expression levels. Data show the mean + SD for n = 3 repeats. ** p
Figure Legend Snippet: The effect of MiR-184 on endogenous Slc25a22. (A) Expression of Slc25a22 mRNA. Quantitative RT-PCR analyses of the expression levels of Slc25a22 mRNA were performed 48 h after transfection of MIN6 cells with miR-184 or control miRNA. The 18S ribosomal RNA was used to normalize the expression levels. Data show the mean + SD for n = 3 repeats. ** p

Techniques Used: Expressing, Quantitative RT-PCR, Transfection

28) Product Images from "The protein kinase C inhibitor, Ro-31-7459, is a potent activator of ERK and JNK MAP kinases in HUVECs and yet inhibits cyclic AMP-stimulated SOCS-3 gene induction through inactivation of the transcription factor c-Jun"

Article Title: The protein kinase C inhibitor, Ro-31-7459, is a potent activator of ERK and JNK MAP kinases in HUVECs and yet inhibits cyclic AMP-stimulated SOCS-3 gene induction through inactivation of the transcription factor c-Jun

Journal: Cellular Signalling

doi: 10.1016/j.cellsig.2012.04.016

Protein kinase C inhibitors block human SOCS-3 gene induction in HUVECs. A). HUVECs were stimulated for 5 h with MG132 (10 μM) in the presence or absence of either a combination of 10 μM forskolin plus 10 μM rolipram (F/R; upper panel ) or 10 μM PMA ( lower panel ) plus the indicated concentrations of the protein kinase C (PKC) inhibitors Ro-31-7549 or GF-109203X. Cell extracts were then prepared and immunoblotted with antibodies to SOCS-3 or β-tubulin as indicated. B). HUVECs were stimulated for 5 h with MG132 (10 μM) in the presence or absence of either F/R ( upper panel ) or 10 μM PMA ( lower panel ) plus the indicated concentrations of the PKC inhibitors Ro-31-7549 or Gö-6983. Cell extracts were then prepared and immunoblotted with antibodies to SOCS-3 or β-tubulin as indicated. C). HUVECs were stimulated for 5 h in the presence or absence of F/R ( upper panel ) or 10 μM PMA ( lower panel ) plus Ro-31-7549 (5 μM), Gö-6983 (25 μM) or GF-109203X (25 μM). Total RNA was then extracted from cells and subjected to one-step RT-PCR, with specific primers towards SOCS-3 or actin, as described in Materials and methods . Amplified DNA fragments were visualised by agarose gel electrophoresis.
Figure Legend Snippet: Protein kinase C inhibitors block human SOCS-3 gene induction in HUVECs. A). HUVECs were stimulated for 5 h with MG132 (10 μM) in the presence or absence of either a combination of 10 μM forskolin plus 10 μM rolipram (F/R; upper panel ) or 10 μM PMA ( lower panel ) plus the indicated concentrations of the protein kinase C (PKC) inhibitors Ro-31-7549 or GF-109203X. Cell extracts were then prepared and immunoblotted with antibodies to SOCS-3 or β-tubulin as indicated. B). HUVECs were stimulated for 5 h with MG132 (10 μM) in the presence or absence of either F/R ( upper panel ) or 10 μM PMA ( lower panel ) plus the indicated concentrations of the PKC inhibitors Ro-31-7549 or Gö-6983. Cell extracts were then prepared and immunoblotted with antibodies to SOCS-3 or β-tubulin as indicated. C). HUVECs were stimulated for 5 h in the presence or absence of F/R ( upper panel ) or 10 μM PMA ( lower panel ) plus Ro-31-7549 (5 μM), Gö-6983 (25 μM) or GF-109203X (25 μM). Total RNA was then extracted from cells and subjected to one-step RT-PCR, with specific primers towards SOCS-3 or actin, as described in Materials and methods . Amplified DNA fragments were visualised by agarose gel electrophoresis.

Techniques Used: Blocking Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis

29) Product Images from "A novel source for miR-21 expression through the alternative polyadenylation of VMP1 gene transcripts"

Article Title: A novel source for miR-21 expression through the alternative polyadenylation of VMP1 gene transcripts

Journal: Nucleic Acids Research

doi: 10.1093/nar/gks308

Phorbol ester mediated induction of pri-miR-21 and VMP1–miR-21. ( A ) Northern blot analysis of total RNA from HL-60 cells treated with PMA (+) or vehicle (−) for 6 h. At the left, probes used for each blot and identifying letters for the transcripts are indicated. ( B ) Quantitative RT-PCR analysis of HL-60 cells treated with PMA or vehicle. Relative induced expression of VMP1 and VMP1–miR-21 (exons 10-11), pri-miR-21 (intron 11) and an area surrounding miR-21 hairpin (miR-21) are normalized to actin. Note that VMP1 and VMP1–miR-21 are indistinguishable by this quantitative RT-PCR analysis. Error bars represent standard deviations derived from two independent experiments measured per triplicate. *** P
Figure Legend Snippet: Phorbol ester mediated induction of pri-miR-21 and VMP1–miR-21. ( A ) Northern blot analysis of total RNA from HL-60 cells treated with PMA (+) or vehicle (−) for 6 h. At the left, probes used for each blot and identifying letters for the transcripts are indicated. ( B ) Quantitative RT-PCR analysis of HL-60 cells treated with PMA or vehicle. Relative induced expression of VMP1 and VMP1–miR-21 (exons 10-11), pri-miR-21 (intron 11) and an area surrounding miR-21 hairpin (miR-21) are normalized to actin. Note that VMP1 and VMP1–miR-21 are indistinguishable by this quantitative RT-PCR analysis. Error bars represent standard deviations derived from two independent experiments measured per triplicate. *** P

Techniques Used: Northern Blot, Quantitative RT-PCR, Expressing, Derivative Assay

Drosha knock-down enhances VMP1 transcript levels. Total RNA was prepared from ( A ) LNCaP, ( B ) MCF-7 or ( C ) LAPC-4 cells, which had been transfected for 72 h with a negative control siRNA (siNeg) or Drosha siRNA (siDrosha). Expression of Drosha and VMP1, relative to actin, was measured by quantitative RT-PCR. Quantification is the result of three independent measurements. * P
Figure Legend Snippet: Drosha knock-down enhances VMP1 transcript levels. Total RNA was prepared from ( A ) LNCaP, ( B ) MCF-7 or ( C ) LAPC-4 cells, which had been transfected for 72 h with a negative control siRNA (siNeg) or Drosha siRNA (siDrosha). Expression of Drosha and VMP1, relative to actin, was measured by quantitative RT-PCR. Quantification is the result of three independent measurements. * P

Techniques Used: Transfection, Negative Control, Expressing, Quantitative RT-PCR

Relative quantification of pri-miR-21 and VMP1–miR-21 transcripts. ( A ) Total RNA from DU-145 and HL-60 cells was specifically pulled-down (RNA pull-down) with a biotinylated probe in the miR-21 hairpin region and control or miR-21-specific RNA was quantified by quantitative RT-PCR. The y -axis represents log fold enrichment after miR-21 pull down, relative to input RNA. ( B ) Total RNA from MCF-7 cells was reverse transcribed using a miR-21 GSP. The resulting cDNA was quantified by qPCR for control or miR-21 transcripts to determine enrichment by GSP reverse transcription. ( C ) RNA from miR-21 pull down in DU-145 and HL-60 cells was reverse transcribed and amplicons specific for VMP1–miR-21 (exons 10-11), pri-miR-21 (intron 11) or both transcripts (exon 12 and miR-21) were quantified by qPCR. Ct values obtained for captured transcripts were referred to values of exon 12 and plotted in a logarithmic scale. ( D ) cDNA from MCF-7 miR-21 GSP reverse transcription was quantified by qPCR for VMP1–miR-21 (exons 10-11), pri-miR-21 (intron 11) or both transcripts (exon 12 and miR-21) amplicons. Ct values obtained for captured transcripts were referred to values of exon 12 and plotted in a logarithmic scale. Error bars represent standard deviations derived from one representative experiment measured per triplicate. Error bars represent standard deviations derived from three independent measurements.
Figure Legend Snippet: Relative quantification of pri-miR-21 and VMP1–miR-21 transcripts. ( A ) Total RNA from DU-145 and HL-60 cells was specifically pulled-down (RNA pull-down) with a biotinylated probe in the miR-21 hairpin region and control or miR-21-specific RNA was quantified by quantitative RT-PCR. The y -axis represents log fold enrichment after miR-21 pull down, relative to input RNA. ( B ) Total RNA from MCF-7 cells was reverse transcribed using a miR-21 GSP. The resulting cDNA was quantified by qPCR for control or miR-21 transcripts to determine enrichment by GSP reverse transcription. ( C ) RNA from miR-21 pull down in DU-145 and HL-60 cells was reverse transcribed and amplicons specific for VMP1–miR-21 (exons 10-11), pri-miR-21 (intron 11) or both transcripts (exon 12 and miR-21) were quantified by qPCR. Ct values obtained for captured transcripts were referred to values of exon 12 and plotted in a logarithmic scale. ( D ) cDNA from MCF-7 miR-21 GSP reverse transcription was quantified by qPCR for VMP1–miR-21 (exons 10-11), pri-miR-21 (intron 11) or both transcripts (exon 12 and miR-21) amplicons. Ct values obtained for captured transcripts were referred to values of exon 12 and plotted in a logarithmic scale. Error bars represent standard deviations derived from one representative experiment measured per triplicate. Error bars represent standard deviations derived from three independent measurements.

Techniques Used: Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Derivative Assay

30) Product Images from "SRC-2 Coactivator Deficiency Decreases Functional Reserve in Response to Pressure Overload of Mouse Heart"

Article Title: SRC-2 Coactivator Deficiency Decreases Functional Reserve in Response to Pressure Overload of Mouse Heart

Journal: PLoS ONE

doi: 10.1371/journal.pone.0053395

Ablation of SRC-2 in the mouse mimics the metabolic gene expression of a stressed heart. A – C , Quantitative PCR analysis (qPCR) of gene expression of the indicated gene involved in glycolytic ( A ), fatty acid ( B ), and Krebs cycle and oxidative phosphorylation ( C ), metabolic pathways. RNA was isolated from WT and SRC-2 KO hearts (n = 5). Individual gene expression is analyzed by ΔΔCt method with 18S RNA expression used as a normalizer and expression relative to WT. D , Lactate levels in WT and SRC-2 KO mouse heart tissue lysates. * = p≤0.05, ** = p≤0.01, and *** = p≤0.001. (Slc2a1- facilitated glucose transporter member 1 (GLUT1) # , Slc2a4- facilitated glucose transporter member 4 (GLUT4) # , Ldhd- lactate hydrogenase isoform d # , Pfkfb1- 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 1 # , Fbp2- fructose 1,6-bisphophatase 2 # , Pkm2- pyruvate kinase, muscle, Pdk2 and 4- pyruvate dehydrogenase kinase 2 # and 4 # , Fabp5, 7, and 1- fatty acid binding proteins 5 # , 7 # , and 1 # , CD36- fatty acid transporter, AcsL1- acyl-CoA synthetase long-chain 1; Dgat 1 and 2- diacylglycerol O-acyltransferase 1 and 2, Acadl- long chain acyl-Coenzyme A dehydrogenase, Idh2- isocitrate dehydrogenase 2, Fh1- fumarate hydratase 1, ATPaf2- ATP synthase mitochondrial F1 complex assembly factor 2, COX4- cytochrome c oxidase subunit IV isoform 1, ATP5b- ATP synthase F1β subunit). A # indicates a gene identified in the microarray.
Figure Legend Snippet: Ablation of SRC-2 in the mouse mimics the metabolic gene expression of a stressed heart. A – C , Quantitative PCR analysis (qPCR) of gene expression of the indicated gene involved in glycolytic ( A ), fatty acid ( B ), and Krebs cycle and oxidative phosphorylation ( C ), metabolic pathways. RNA was isolated from WT and SRC-2 KO hearts (n = 5). Individual gene expression is analyzed by ΔΔCt method with 18S RNA expression used as a normalizer and expression relative to WT. D , Lactate levels in WT and SRC-2 KO mouse heart tissue lysates. * = p≤0.05, ** = p≤0.01, and *** = p≤0.001. (Slc2a1- facilitated glucose transporter member 1 (GLUT1) # , Slc2a4- facilitated glucose transporter member 4 (GLUT4) # , Ldhd- lactate hydrogenase isoform d # , Pfkfb1- 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 1 # , Fbp2- fructose 1,6-bisphophatase 2 # , Pkm2- pyruvate kinase, muscle, Pdk2 and 4- pyruvate dehydrogenase kinase 2 # and 4 # , Fabp5, 7, and 1- fatty acid binding proteins 5 # , 7 # , and 1 # , CD36- fatty acid transporter, AcsL1- acyl-CoA synthetase long-chain 1; Dgat 1 and 2- diacylglycerol O-acyltransferase 1 and 2, Acadl- long chain acyl-Coenzyme A dehydrogenase, Idh2- isocitrate dehydrogenase 2, Fh1- fumarate hydratase 1, ATPaf2- ATP synthase mitochondrial F1 complex assembly factor 2, COX4- cytochrome c oxidase subunit IV isoform 1, ATP5b- ATP synthase F1β subunit). A # indicates a gene identified in the microarray.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Isolation, RNA Expression, Binding Assay, Microarray

Shifts in sarcomeric and stress response gene expression profiles of SRC-2 KO. A–B , qPCR analysis of the indicated actin, myosin, and tubulin isoforms ( A ), and cardiac stress response ( B ) genes. RNA was isolated from WT and SRC-2 KO hearts (n = 5). Individual gene expression is analyzed by ΔΔCt method with 18S RNA expression used as a normalizer and expression relative to WT. * = p≤0.05 and ** = p≤0.01. (Actc1- cardiac actin, Acta2- smooth muscle actin # , Myh-myosin heavy chain 6 and 7, Mlc- myosin light chain (Myl2v), Tppp3- tubulin polymerization promoting protein family member 3 # , Tuba8- tubulin, alpha 8 # , c-myc- myelocytometosis oncogene, ANF- atrial natiuretic factor, Serca 2- cardiac muscle Ca+2 transporting ATPase). A # indicates a gene identified in the microarray.
Figure Legend Snippet: Shifts in sarcomeric and stress response gene expression profiles of SRC-2 KO. A–B , qPCR analysis of the indicated actin, myosin, and tubulin isoforms ( A ), and cardiac stress response ( B ) genes. RNA was isolated from WT and SRC-2 KO hearts (n = 5). Individual gene expression is analyzed by ΔΔCt method with 18S RNA expression used as a normalizer and expression relative to WT. * = p≤0.05 and ** = p≤0.01. (Actc1- cardiac actin, Acta2- smooth muscle actin # , Myh-myosin heavy chain 6 and 7, Mlc- myosin light chain (Myl2v), Tppp3- tubulin polymerization promoting protein family member 3 # , Tuba8- tubulin, alpha 8 # , c-myc- myelocytometosis oncogene, ANF- atrial natiuretic factor, Serca 2- cardiac muscle Ca+2 transporting ATPase). A # indicates a gene identified in the microarray.

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

Loss of SRC-2 results in decreased expression of several cardiac transcription factors important for controlling metabolic and sarcomeric gene expression. A–D and F , qPCR analysis of the indicated transcription factor and transcription co-activator genes. RNA was isolated from WT and SRC-2 KO hearts (n = 5). Individual gene expression is analyzed by ΔΔCt method with 18S RNA expression used as a normalizer and expression relative to WT. E , Immunoblot for GATA-4 and MEF2 protein expression in WT and SRC-2 KO heart tissue lysates (n = 3).* = p≤0.05, ** = p≤0.01, and *** = p≤0.001. (PPAR- peroxisome proliferator activated-receptor α, β/δ, and γ, PGC-1- peroxisome proliferator activated-receptor γ coactivator-1 α and β, SRC- steroid receptor coactivator 1 and 3, SRF- serum response factor, GATA- GATA binding protein 4 and 6, MEF2c- myocyte enhancer factor 2c, Hand 2- heart and neural crest derivatives 2, Nkx2.5- NK2 transcription factor related 5, Gfat- glutamine frustose-6-phosphate transaminase 1 and 2, Tbx5- T-box 5 # , NRF1- nuclear respiratory factor 1, ERR- estrogen-related receptor α). A # indicates a gene identified in the microarray.
Figure Legend Snippet: Loss of SRC-2 results in decreased expression of several cardiac transcription factors important for controlling metabolic and sarcomeric gene expression. A–D and F , qPCR analysis of the indicated transcription factor and transcription co-activator genes. RNA was isolated from WT and SRC-2 KO hearts (n = 5). Individual gene expression is analyzed by ΔΔCt method with 18S RNA expression used as a normalizer and expression relative to WT. E , Immunoblot for GATA-4 and MEF2 protein expression in WT and SRC-2 KO heart tissue lysates (n = 3).* = p≤0.05, ** = p≤0.01, and *** = p≤0.001. (PPAR- peroxisome proliferator activated-receptor α, β/δ, and γ, PGC-1- peroxisome proliferator activated-receptor γ coactivator-1 α and β, SRC- steroid receptor coactivator 1 and 3, SRF- serum response factor, GATA- GATA binding protein 4 and 6, MEF2c- myocyte enhancer factor 2c, Hand 2- heart and neural crest derivatives 2, Nkx2.5- NK2 transcription factor related 5, Gfat- glutamine frustose-6-phosphate transaminase 1 and 2, Tbx5- T-box 5 # , NRF1- nuclear respiratory factor 1, ERR- estrogen-related receptor α). A # indicates a gene identified in the microarray.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Isolation, RNA Expression, Pyrolysis Gas Chromatography, Binding Assay, Microarray

31) Product Images from "Microarray and deep sequencing cross-platform analysis of the mirRNome and isomiR variation in response to epidermal growth factor"

Article Title: Microarray and deep sequencing cross-platform analysis of the mirRNome and isomiR variation in response to epidermal growth factor

Journal: BMC Genomics

doi: 10.1186/1471-2164-14-371

RT-qPCR validation of EGF-regulated miRNAs. Total RNA prepared from cells lysates were analyzed by quantitative real time PCR using the miRCURY LNA™ microRNA PCR System (Exiqon) for each of the miRNAs as indicated. A . Control treatment without inhibitors: HeLa cells were serum-starved for 24 hours and treated with EGF for 1 and 6 hours. B . Inhibitor treatment. HeLa cells were serum-starved for 24 hours and treated with EGF for 6 hours in the presence or absence of protein kinase inhibitors: AG1470 (EGFR inhibitor), U0126 (MEK inhibitor) and Wortmannin (PI3K inhibitor). In addition, HeLa cells were transfected with a constitutively active form of Ras (RasV12). Effective pathway inhibition was verified by western blotting in parallel samples from the same experiment (see Additional file 8 ).
Figure Legend Snippet: RT-qPCR validation of EGF-regulated miRNAs. Total RNA prepared from cells lysates were analyzed by quantitative real time PCR using the miRCURY LNA™ microRNA PCR System (Exiqon) for each of the miRNAs as indicated. A . Control treatment without inhibitors: HeLa cells were serum-starved for 24 hours and treated with EGF for 1 and 6 hours. B . Inhibitor treatment. HeLa cells were serum-starved for 24 hours and treated with EGF for 6 hours in the presence or absence of protein kinase inhibitors: AG1470 (EGFR inhibitor), U0126 (MEK inhibitor) and Wortmannin (PI3K inhibitor). In addition, HeLa cells were transfected with a constitutively active form of Ras (RasV12). Effective pathway inhibition was verified by western blotting in parallel samples from the same experiment (see Additional file 8 ).

Techniques Used: Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Transfection, Inhibition, Western Blot

32) Product Images from "RNAi Induces Innate Immunity through Multiple Cellular Signaling Pathways"

Article Title: RNAi Induces Innate Immunity through Multiple Cellular Signaling Pathways

Journal: PLoS ONE

doi: 10.1371/journal.pone.0064708

Inhibition of siRNA blocked the siWHx-mediated induction of IFN-β, MxA, and IP-10. PMHs isolated from WHV Tg mice were transfected with 100 nM of siWHx or cotransfected with 100 nM of siWHx and 100 nM of siWHx specific inhibitor (2′-O-methyl antisense RNA), and the mRNA levels of WHV and mouse IFN-β, MxA, and IP-10 were determined by real-time RT-PCR 48 h posttransfection.
Figure Legend Snippet: Inhibition of siRNA blocked the siWHx-mediated induction of IFN-β, MxA, and IP-10. PMHs isolated from WHV Tg mice were transfected with 100 nM of siWHx or cotransfected with 100 nM of siWHx and 100 nM of siWHx specific inhibitor (2′-O-methyl antisense RNA), and the mRNA levels of WHV and mouse IFN-β, MxA, and IP-10 were determined by real-time RT-PCR 48 h posttransfection.

Techniques Used: Inhibition, Isolation, Mouse Assay, Transfection, Quantitative RT-PCR

33) Product Images from "The synthetic triterpenoid, RTA405, increases glomerular filtration rate and reduces angiotensin II-induced contraction of glomerular mesangial cells"

Article Title: The synthetic triterpenoid, RTA405, increases glomerular filtration rate and reduces angiotensin II-induced contraction of glomerular mesangial cells

Journal: Kidney international

doi: 10.1038/ki.2012.393

Bardoxolone methyl and RTA 405 induced expression of Nrf2 target genes in cultured MCs Human MCs (A) and rat mesangial cells (B) were treated with the indicated concentrations of bardoxolone methyl or RTA 405 for 16 hours. Cells were harvested for RNA isolation and cDNA synthesis, and Nrf2 target gene expression was evaluated by real-time PCR. Fold-change values (relative to DMSO-treated samples) are the average of three independent experiments. Nqo1: NAD(P)H dehydrogenase quinone 1; Hmox1: heme oxygenase 1; Gclc: glutamate-cysteine ligase, catalytic subunit; Txnrd1: thioredoxin reductase 1. Error bars represent standard deviation.error of the mean. * P
Figure Legend Snippet: Bardoxolone methyl and RTA 405 induced expression of Nrf2 target genes in cultured MCs Human MCs (A) and rat mesangial cells (B) were treated with the indicated concentrations of bardoxolone methyl or RTA 405 for 16 hours. Cells were harvested for RNA isolation and cDNA synthesis, and Nrf2 target gene expression was evaluated by real-time PCR. Fold-change values (relative to DMSO-treated samples) are the average of three independent experiments. Nqo1: NAD(P)H dehydrogenase quinone 1; Hmox1: heme oxygenase 1; Gclc: glutamate-cysteine ligase, catalytic subunit; Txnrd1: thioredoxin reductase 1. Error bars represent standard deviation.error of the mean. * P

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

34) Product Images from "c-KIT signaling is targeted by pathogenic Yersinia to suppress the host immune response"

Article Title: c-KIT signaling is targeted by pathogenic Yersinia to suppress the host immune response

Journal: BMC Microbiology

doi: 10.1186/1471-2180-13-249

Pathogenic Yersinia requires c-KIT activity for suppression of transcription factor and pro-inflammatory cytokine expression. (A-B) Analysis of signal transduction pathways in Y. pestis -infected THP1 cells in absence of c-KIT. THP1 cells untreated or pre-treated with 1μM OSI-930 for 18 h were infected with Y. pestis Ind195 at MOI 20 for 1h. RNA was isolated, converted to cDNA, and applied to a RT Profiler PCR Array for human signal transduction pathway expression analysis. Dot plots compare gene expression profiles between uninfected THP-1 cells and (A) Y. pestis Ind195-infected THP-1 cells or ( B ) OSI-930-pretreated, Y. pestis Ind195 infected THP-1 cells. Genes that do not exhibit significant expression changes between the control and experimental samples are concentrated between the two black lines. The black lines define the assay cut-off of 3-fold induction or 70% reduction of transcript levels. Genes of interest are highlighted in black. (C) Inhibition of c-KIT recovers EGR1, chemokine, and cell adhesion transcript levels in pathogenic Yersinia -infected THP1 cells. THP1 cells were pre-treated with 1μM OSI-930 for 18 h or were left untreated prior to infection with Y. pestis Ind195 at MOI 10 for 1 h. EGR1, VCAM1, CCL20, and IL-8 mRNA levels were determined by Taqman qPCR using total RNA isolated 24 h post-infection. Depicted RNA levels are relative to untreated THP1 control samples and were calculated using the 2 -ΔΔCt formula. A ‘*” denotes that relative RNA levels were significantly different (p
Figure Legend Snippet: Pathogenic Yersinia requires c-KIT activity for suppression of transcription factor and pro-inflammatory cytokine expression. (A-B) Analysis of signal transduction pathways in Y. pestis -infected THP1 cells in absence of c-KIT. THP1 cells untreated or pre-treated with 1μM OSI-930 for 18 h were infected with Y. pestis Ind195 at MOI 20 for 1h. RNA was isolated, converted to cDNA, and applied to a RT Profiler PCR Array for human signal transduction pathway expression analysis. Dot plots compare gene expression profiles between uninfected THP-1 cells and (A) Y. pestis Ind195-infected THP-1 cells or ( B ) OSI-930-pretreated, Y. pestis Ind195 infected THP-1 cells. Genes that do not exhibit significant expression changes between the control and experimental samples are concentrated between the two black lines. The black lines define the assay cut-off of 3-fold induction or 70% reduction of transcript levels. Genes of interest are highlighted in black. (C) Inhibition of c-KIT recovers EGR1, chemokine, and cell adhesion transcript levels in pathogenic Yersinia -infected THP1 cells. THP1 cells were pre-treated with 1μM OSI-930 for 18 h or were left untreated prior to infection with Y. pestis Ind195 at MOI 10 for 1 h. EGR1, VCAM1, CCL20, and IL-8 mRNA levels were determined by Taqman qPCR using total RNA isolated 24 h post-infection. Depicted RNA levels are relative to untreated THP1 control samples and were calculated using the 2 -ΔΔCt formula. A ‘*” denotes that relative RNA levels were significantly different (p

Techniques Used: Activity Assay, Expressing, Transduction, Infection, Isolation, Polymerase Chain Reaction, Inhibition, Real-time Polymerase Chain Reaction

35) Product Images from "Evolution of GOLDEN2-LIKE gene function in C3 and C4 plants"

Article Title: Evolution of GOLDEN2-LIKE gene function in C3 and C4 plants

Journal: Planta

doi: 10.1007/s00425-012-1754-3

Analysis of GLK gene expression in leaves of Sorghum bicolor and Cleome gynandra . a Gel blot of 10 µg total RNA extracted from total leaf (T), mesophyll (M) or bundle sheath (BS) cells of sorghum. The blot was hybridized with SbGLK1 and SbGLK2, and with maize PEPC (M cell-specific) and RbcS (BS cell-specific) sequences to confirm the purity of the cell preparations. Ethidium bromide stained ribosomal RNA bands are shown as loading controls. b Transcript levels of SbGLK1 and SbGLK2 in BS and M cells of sorghum as determined by 40 bp Illumina RNA sequencing, and quantified as reads per million (RPM) to two decimal places. Sorghum PEPC (M cell-specific) and NADP - ME (BS cell-specific) transcript levels demonstrate the purity of the cell extracts. Significance of differential gene expression between M and BS samples was calculated as described in the “ Results ”. c Bootstrapped maximum likelihood phylogenetic tree of a subset of GLK genes from the Brassicales with the Aquilegia (Ranunculales) gene used as the outgroup. d qPCR of CgGLK1 and CgGLK2 with RNA extracted from C. gynandra M and BS cells separated by LCM. Values are shown relative to Actin7 transcript levels. Bars and error bars represent means and standard errors of three biological replicates, respectively. e Log2 of the ratio of BS/M transcript levels as determined by qPCR as in c . CgPPC2 (M cell-specific) and CgNADME2 (BS cell-specific) ratios confirm the purity of the cell preparations
Figure Legend Snippet: Analysis of GLK gene expression in leaves of Sorghum bicolor and Cleome gynandra . a Gel blot of 10 µg total RNA extracted from total leaf (T), mesophyll (M) or bundle sheath (BS) cells of sorghum. The blot was hybridized with SbGLK1 and SbGLK2, and with maize PEPC (M cell-specific) and RbcS (BS cell-specific) sequences to confirm the purity of the cell preparations. Ethidium bromide stained ribosomal RNA bands are shown as loading controls. b Transcript levels of SbGLK1 and SbGLK2 in BS and M cells of sorghum as determined by 40 bp Illumina RNA sequencing, and quantified as reads per million (RPM) to two decimal places. Sorghum PEPC (M cell-specific) and NADP - ME (BS cell-specific) transcript levels demonstrate the purity of the cell extracts. Significance of differential gene expression between M and BS samples was calculated as described in the “ Results ”. c Bootstrapped maximum likelihood phylogenetic tree of a subset of GLK genes from the Brassicales with the Aquilegia (Ranunculales) gene used as the outgroup. d qPCR of CgGLK1 and CgGLK2 with RNA extracted from C. gynandra M and BS cells separated by LCM. Values are shown relative to Actin7 transcript levels. Bars and error bars represent means and standard errors of three biological replicates, respectively. e Log2 of the ratio of BS/M transcript levels as determined by qPCR as in c . CgPPC2 (M cell-specific) and CgNADME2 (BS cell-specific) ratios confirm the purity of the cell preparations

Techniques Used: Expressing, Western Blot, Staining, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Laser Capture Microdissection

36) Product Images from "Upregulated Genes In Sporadic, Idiopathic Pulmonary Arterial Hypertension"

Article Title: Upregulated Genes In Sporadic, Idiopathic Pulmonary Arterial Hypertension

Journal: Respiratory Research

doi: 10.1186/1465-9921-7-1

Localisation of TIMP-3 in IPAH lung tissue. Immunocytochemistry for TIMP-3 was carried out using an polyclonal antisera raised against the human carboxy-terminal peptide of TIMP-3. Plexiform lesion showing location of TIMP-3 in subendothelium. (A). Hypertrophied artery, showing location of TIMP-3 in in vascular smooth muscle cells and myoblasts (B). Scale bars = 100 μm. Protein extracted from IPAH and donor lung tissues was analysed by western blot using an antiserum to TIMP-3 (C). The antibody recognises both the unglycosalated form (24 kDa), and the glycosalated form of TIMP-3 (30 kDa). Expression of TIMP-3 in human cells (D). RNA extracted from human cell lines and explant derived cells was reverse transcribed and amplified by PCR and run on agarose/ethidium bromide stained gels. RT-PCR for TIMP-3 (top panel) and G3PDH (bottom panel). The cell types examined were: Lanes; 1, HL60 (promyelocytic leukaemia); 2, Daudi (Burkitt's lymphoma); 3, EB-transformed lymphocytes; 4, K562 (erythroleukemia) 5, pulmonary adult fibroblasts; 6, pulmonary artery smooth muscle; 7, bronchial smooth muscle; 8, A549 (adenocarcinoma alveolar epithelial); 9, H322 (adenocarcinoma bronchial epithelial); 10, placental microvascular endothelial; 11, umbilical vein endothelial; -, no cDNA negative control; + control lung cDNA; M, phiX174 DNA/HaeIII markers.
Figure Legend Snippet: Localisation of TIMP-3 in IPAH lung tissue. Immunocytochemistry for TIMP-3 was carried out using an polyclonal antisera raised against the human carboxy-terminal peptide of TIMP-3. Plexiform lesion showing location of TIMP-3 in subendothelium. (A). Hypertrophied artery, showing location of TIMP-3 in in vascular smooth muscle cells and myoblasts (B). Scale bars = 100 μm. Protein extracted from IPAH and donor lung tissues was analysed by western blot using an antiserum to TIMP-3 (C). The antibody recognises both the unglycosalated form (24 kDa), and the glycosalated form of TIMP-3 (30 kDa). Expression of TIMP-3 in human cells (D). RNA extracted from human cell lines and explant derived cells was reverse transcribed and amplified by PCR and run on agarose/ethidium bromide stained gels. RT-PCR for TIMP-3 (top panel) and G3PDH (bottom panel). The cell types examined were: Lanes; 1, HL60 (promyelocytic leukaemia); 2, Daudi (Burkitt's lymphoma); 3, EB-transformed lymphocytes; 4, K562 (erythroleukemia) 5, pulmonary adult fibroblasts; 6, pulmonary artery smooth muscle; 7, bronchial smooth muscle; 8, A549 (adenocarcinoma alveolar epithelial); 9, H322 (adenocarcinoma bronchial epithelial); 10, placental microvascular endothelial; 11, umbilical vein endothelial; -, no cDNA negative control; + control lung cDNA; M, phiX174 DNA/HaeIII markers.

Techniques Used: Immunocytochemistry, Western Blot, Expressing, Derivative Assay, Amplification, Polymerase Chain Reaction, Staining, Reverse Transcription Polymerase Chain Reaction, Transformation Assay, Negative Control

37) Product Images from "Genome-Wide DNA Methylation Profiling in Cultured Eutopic and Ectopic Endometrial Stromal Cells"

Article Title: Genome-Wide DNA Methylation Profiling in Cultured Eutopic and Ectopic Endometrial Stromal Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0083612

The mRNA levels of NR5A1 (A), STAR (B), STRA6 (C) and HSD17B2 (D) in ESCa, choESC, eutopic endometrium and chocolate cysts. ESCa (n = 7), choESC (n = 5), eutopic endometria (n = 17) and chocolate cysts (n = 6) were subjected to total RNA isolation followed by real-time RT-PCR. The relative mRNA expression normalized to that of TBP (an internal control) was calculated. The values are the means ±SD. *, p
Figure Legend Snippet: The mRNA levels of NR5A1 (A), STAR (B), STRA6 (C) and HSD17B2 (D) in ESCa, choESC, eutopic endometrium and chocolate cysts. ESCa (n = 7), choESC (n = 5), eutopic endometria (n = 17) and chocolate cysts (n = 6) were subjected to total RNA isolation followed by real-time RT-PCR. The relative mRNA expression normalized to that of TBP (an internal control) was calculated. The values are the means ±SD. *, p

Techniques Used: Isolation, Quantitative RT-PCR, Expressing

38) Product Images from "Genome-Wide Effects of Selenium and Translational Uncoupling on Transcription in the Termite Gut Symbiont Treponema primitia"

Article Title: Genome-Wide Effects of Selenium and Translational Uncoupling on Transcription in the Termite Gut Symbiont Treponema primitia

Journal: mBio

doi: 10.1128/mBio.00869-13

RNA-Seq transcriptional maps of T. primitia genes show a variety of transcriptional responses to translation inhibition. The cultures analyzed were the same as in Fig. 3. The three cultures were initially grown without selenium and were subsequently amended with selenium alone (50 nM sodium selenite) 6 h prior to sampling (blue line) or selenium and tetracycline concurrently 6 h prior to sampling (red line). The control culture (gray line) received neither treatment. Traces in each panel show the moving average of Illumina RNA sequencing read depth over a 200-bp sliding window of nucleotide positions. The black arrow on the x axis of each panel indicates the open reading frame of the gene. (A to C) Examples of genes for which transcription is truncated in the culture treated with tetracycline compared to other conditions. Putative gene products are ATP-dependent Clp protease (ATP-binding subunit, ClpA) (A), pyruvate phosphate dikinase (B), and extracellular solute-binding protein (C). (D to F) Examples of genes for which transcription is similar in the culture treated with tetracycline to other conditions. Putative identities are TPR (tetratrico peptide repeat) domain protein (D), CTP synthetase (E), and multiple sugar ABC transporter (substrate-binding subunit) (F). (G to I) Examples of genes for which transcript levels are higher over the length of the gene in the culture treated with tetracycline than under other conditions. Putative identities are cytidylate kinase (G), methyl corrinoid protein (H), and phosphate acetyltransferase (I).
Figure Legend Snippet: RNA-Seq transcriptional maps of T. primitia genes show a variety of transcriptional responses to translation inhibition. The cultures analyzed were the same as in Fig. 3. The three cultures were initially grown without selenium and were subsequently amended with selenium alone (50 nM sodium selenite) 6 h prior to sampling (blue line) or selenium and tetracycline concurrently 6 h prior to sampling (red line). The control culture (gray line) received neither treatment. Traces in each panel show the moving average of Illumina RNA sequencing read depth over a 200-bp sliding window of nucleotide positions. The black arrow on the x axis of each panel indicates the open reading frame of the gene. (A to C) Examples of genes for which transcription is truncated in the culture treated with tetracycline compared to other conditions. Putative gene products are ATP-dependent Clp protease (ATP-binding subunit, ClpA) (A), pyruvate phosphate dikinase (B), and extracellular solute-binding protein (C). (D to F) Examples of genes for which transcription is similar in the culture treated with tetracycline to other conditions. Putative identities are TPR (tetratrico peptide repeat) domain protein (D), CTP synthetase (E), and multiple sugar ABC transporter (substrate-binding subunit) (F). (G to I) Examples of genes for which transcript levels are higher over the length of the gene in the culture treated with tetracycline than under other conditions. Putative identities are cytidylate kinase (G), methyl corrinoid protein (H), and phosphate acetyltransferase (I).

Techniques Used: RNA Sequencing Assay, Inhibition, Sampling, Binding Assay

39) Product Images from "Foxc1 is required by pericytes during fetal brain angiogenesis"

Article Title: Foxc1 is required by pericytes during fetal brain angiogenesis

Journal: Biology Open

doi: 10.1242/bio.20135009

Vascular defects and brain micro-hemorrhage in pericyte conditional Foxc1 mutants. ( A ) RT-PCR of Foxc1 transcript from three RNA sources: (1) E18.5 PDGFrb-cre ; Foxc1fl-+ meninges (2) E18.5 microvessels from PDGFrb-cre ; Foxc1fl-+ brain and (3) E18.5 microvessels from PDGFrb-cre ; Foxc1fl-fl brain. Housekeeping gene GAPDH serves as internal control. ( B ) Whole embryo images of E18.5 PDGFrβ-cre ; Foxc1fl-fl mutant and PDGFrb-cre ; Foxc1fl-+ littermate. ( C–E ) Dorsal view of E18.5 PDGFrβ-cre ; Foxc1fl-+ (C) and PDGFrβ-cre ; Foxc1fl-fl mutant (D,E) brains. Arrows indicate hemorrhage within the cerebral cortex. ( F , G ) Glut-1 (green), Ib4 (red) and DAPI (blue) stained cortical sections of E18.5 PDGFrβ-cre ; Foxc1fl-fl mutant and PDGFrb-cre ; Foxc1fl-+ animals. ( H ) Graphs depicting quantification of average cerebral vessel diameter (top) and cerebral vessel density (bottom) in E18.5 PDGFrβ-cre ; Foxc1fl-fl mutant and PDGFrβ-cre ; Foxc1fl-+ animals. Means represent analysis from three separate litters ( n = 3). ( I , J ) ICAM-1 and IB4 immunofluorescence of E18.5 PDGFrβ-cre ; Foxc1fl-+ and PDGFrβ-cre ; Foxc1fl-fl cortices. Arrowheads indicate superficial ICAM-1+ cerebral vessels descending from the perineural vascular plexus (PNVP). Arrows indicate deeper, ICAM-1+ vessels in PDGFrβ-cre ; Foxc1fl-fl mutant cortices. ( K ) Quantification of percentage of ICAM+ cerebral vessels in control and mutant samples analyzed from three independent litters ( n = 3). ( L , M ) Sagittal view of midbrain and cerebellum of E18.5 PDGFrβ-cre ; Foxc1fl-fl mutant and PDGFrb-cre ; Foxc1fl-+ brains. Arrows indicate hemorrhage sites in PDGFrβ-cre ; Foxc1fl-fl sample. ( N , O ) Glut-1 (green), Ib4 (red), DAPI (blue) immunofluorescence at the level of the midbrain (N) and cerebellum (O) in E18.5 PDGFrβ-cre ; Foxc1fl-fl mutant brains. In G,N,O, arrowheads indicate amoeboid morphology of Ib4+ activated microglia; arrows indicate dilated/dysplastic vessels; asterisks indicate red blood cells in the neural parenchyma. Scale bars: 100 µm. Asterisks in graph indicate a statistically significant difference from PDGFrβ-cre ; Foxc1fl-+ samples ( P
Figure Legend Snippet: Vascular defects and brain micro-hemorrhage in pericyte conditional Foxc1 mutants. ( A ) RT-PCR of Foxc1 transcript from three RNA sources: (1) E18.5 PDGFrb-cre ; Foxc1fl-+ meninges (2) E18.5 microvessels from PDGFrb-cre ; Foxc1fl-+ brain and (3) E18.5 microvessels from PDGFrb-cre ; Foxc1fl-fl brain. Housekeeping gene GAPDH serves as internal control. ( B ) Whole embryo images of E18.5 PDGFrβ-cre ; Foxc1fl-fl mutant and PDGFrb-cre ; Foxc1fl-+ littermate. ( C–E ) Dorsal view of E18.5 PDGFrβ-cre ; Foxc1fl-+ (C) and PDGFrβ-cre ; Foxc1fl-fl mutant (D,E) brains. Arrows indicate hemorrhage within the cerebral cortex. ( F , G ) Glut-1 (green), Ib4 (red) and DAPI (blue) stained cortical sections of E18.5 PDGFrβ-cre ; Foxc1fl-fl mutant and PDGFrb-cre ; Foxc1fl-+ animals. ( H ) Graphs depicting quantification of average cerebral vessel diameter (top) and cerebral vessel density (bottom) in E18.5 PDGFrβ-cre ; Foxc1fl-fl mutant and PDGFrβ-cre ; Foxc1fl-+ animals. Means represent analysis from three separate litters ( n = 3). ( I , J ) ICAM-1 and IB4 immunofluorescence of E18.5 PDGFrβ-cre ; Foxc1fl-+ and PDGFrβ-cre ; Foxc1fl-fl cortices. Arrowheads indicate superficial ICAM-1+ cerebral vessels descending from the perineural vascular plexus (PNVP). Arrows indicate deeper, ICAM-1+ vessels in PDGFrβ-cre ; Foxc1fl-fl mutant cortices. ( K ) Quantification of percentage of ICAM+ cerebral vessels in control and mutant samples analyzed from three independent litters ( n = 3). ( L , M ) Sagittal view of midbrain and cerebellum of E18.5 PDGFrβ-cre ; Foxc1fl-fl mutant and PDGFrb-cre ; Foxc1fl-+ brains. Arrows indicate hemorrhage sites in PDGFrβ-cre ; Foxc1fl-fl sample. ( N , O ) Glut-1 (green), Ib4 (red), DAPI (blue) immunofluorescence at the level of the midbrain (N) and cerebellum (O) in E18.5 PDGFrβ-cre ; Foxc1fl-fl mutant brains. In G,N,O, arrowheads indicate amoeboid morphology of Ib4+ activated microglia; arrows indicate dilated/dysplastic vessels; asterisks indicate red blood cells in the neural parenchyma. Scale bars: 100 µm. Asterisks in graph indicate a statistically significant difference from PDGFrβ-cre ; Foxc1fl-+ samples ( P

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Staining, Immunofluorescence

40) Product Images from "Loss of Catalytically Inactive Lipid Phosphatase Myotubularin-related Protein 12 Impairs Myotubularin Stability and Promotes Centronuclear Myopathy in Zebrafish"

Article Title: Loss of Catalytically Inactive Lipid Phosphatase Myotubularin-related Protein 12 Impairs Myotubularin Stability and Promotes Centronuclear Myopathy in Zebrafish

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1003583

Expression patterns and morpholino-based knockdown of mtmr12 in developing zebrafish. (A) Whole mount in-situ hybridization detected ubiquitous expression of mtmr12 and mtm1 transcripts in zebrafish embryos at 1 dpf (above). Below is RT-PCR analysis of mtm1 and mtmr12 expression during zebrafish development using RNA extracts from whole zebrafish embryos at indicated developmental timepoints. (B) Synergistic expression level of Mtm1 and Mtmr12 transcripts and protein at indicated time points of C2C12 differentiation (0–9 days) monitored by RT-quantitative PCR (corresponding histogram, *P≤0.05) and by western blot analysis (right panel). (C) Live embryos at 3 dpf injected with control, mtmr12 , mtm1 or both mtmr12 and mtm1 morpholinos in normal (left) and polarized lights (right). mtmr12 morphant fish showed a dorsal curvature in skeletal muscle and reduced birefringence in polarized light similar to mtm1 morphant embryos. mtmr12 morphant fish also exhibited pericardial edema (arrow). mtmr12-mtm1 double knockdown fish exhibited smaller size and reduced birefringence relative to mtm1 or mtmr12 alone morphant fish. (D) mtmr12 mRNA levels in mtmr12 morphant zebrafish following injection of two different amounts of morpholino (indicated below, upper panel). In mtm1 morphant fish, no residual myotubularin was observed showing that mtm1 morpholinos are completely penetrant to the limits of detection for western blotting. (E) Over-expression of human MTMR12 mRNA rescued small body length and skeletal muscle abnormalities observed in mtmr12 morphant embryos. (F) Quantification of the chorion hatching at 60 hpf. The number of embryos was quantified in three independent clutches (number of embryos in each clutch = 90–120). (G) Quantification of touch evoke response at 3 dpf (n = 5–8 embryos were assayed in each morpholino group).* P ≤0.01.
Figure Legend Snippet: Expression patterns and morpholino-based knockdown of mtmr12 in developing zebrafish. (A) Whole mount in-situ hybridization detected ubiquitous expression of mtmr12 and mtm1 transcripts in zebrafish embryos at 1 dpf (above). Below is RT-PCR analysis of mtm1 and mtmr12 expression during zebrafish development using RNA extracts from whole zebrafish embryos at indicated developmental timepoints. (B) Synergistic expression level of Mtm1 and Mtmr12 transcripts and protein at indicated time points of C2C12 differentiation (0–9 days) monitored by RT-quantitative PCR (corresponding histogram, *P≤0.05) and by western blot analysis (right panel). (C) Live embryos at 3 dpf injected with control, mtmr12 , mtm1 or both mtmr12 and mtm1 morpholinos in normal (left) and polarized lights (right). mtmr12 morphant fish showed a dorsal curvature in skeletal muscle and reduced birefringence in polarized light similar to mtm1 morphant embryos. mtmr12 morphant fish also exhibited pericardial edema (arrow). mtmr12-mtm1 double knockdown fish exhibited smaller size and reduced birefringence relative to mtm1 or mtmr12 alone morphant fish. (D) mtmr12 mRNA levels in mtmr12 morphant zebrafish following injection of two different amounts of morpholino (indicated below, upper panel). In mtm1 morphant fish, no residual myotubularin was observed showing that mtm1 morpholinos are completely penetrant to the limits of detection for western blotting. (E) Over-expression of human MTMR12 mRNA rescued small body length and skeletal muscle abnormalities observed in mtmr12 morphant embryos. (F) Quantification of the chorion hatching at 60 hpf. The number of embryos was quantified in three independent clutches (number of embryos in each clutch = 90–120). (G) Quantification of touch evoke response at 3 dpf (n = 5–8 embryos were assayed in each morpholino group).* P ≤0.01.

Techniques Used: Expressing, In Situ Hybridization, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Western Blot, Injection, Fluorescence In Situ Hybridization, Over Expression

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    Qiagen qrt pcr rna
    Ro 25–6981 Mechanistic Studies Reveal UPR Modulation in Jimpy (A) <t>qRT-PCR</t> of Plp1 for wild-type and jimpy at day 1 of oligodendrocyte differentiation with indicated treatment. Vehicle-treated controls same as Figure S6 D. For Atf6 n = 3 technical replicates per sample. n = 4 technical replicates per sample. (B) qRT-PCR of UPR-related panel for wild-type and jimpy at day 1 of oligodendrocyte differentiation with indicated treatment. Vehicle-treated controls same as Figure S6 D. For Atf6 n = 3 technical replicates per sample. For all other probes n = 4 technical replicates per sample. Error bars represent mean ± SD. See also Figure S6 and Table S6 .
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    <t>Yy1−/−HSCs</t> Exhibit an Aberrant Genetic Network with Corruption of Genes Involved in Cell-Cycle Regulation <t>$$PARABREAKHERE$$RNA-seq-based</t> comparison of gene expression in Yy1+/+ and Yy1−/−sorted HSCs. (A) Heatmap depicting statistically significant, differentially expressed upregulated and downregulated genes. (B) Gene Ontology analysis of genes upregulated in Yy1 −/− HSCs. The most enriched biological processes are shown with corresponding FDR values. F, FDR. (C) GSEA shows that genes involved in cell-cycle progression and G0 to G1 phase entry are enriched in Yy1 −/− HSCs. (D) Relative mRNA expression of genes in Lin − GFP + bone marrow cells of Yy1 +/+ versus Yy1 −/− . (E) Relative mRNA expressions of genes in Lin − GFP + bone marrow cells of Yy1 +/+ + MigR1, Yy1 −/− + YY1, or Yy1 −/− + ΔREPO. Graphs show means ± SEM; *p
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    Antibiotic treatments alter fungal microbiota before and during colitis. a Fungal levels on day 0 in the fecal microbiota of mice treated with vancomycin, colistin, and vehicle were quantified using 18S rRNA <t>qRT-PCR</t> and were normalized to the bacterial population and quantity of DNA (μg of DNA). Data are presented as the mean ± s.e.m. b Shannon index describing the alpha diversity of the fungal microbiota (ITS2) in the fecal microbiota of mice treated with vancomycin (V), colistin (C), and vehicle (H 2 O). c Beta diversity. Principal coordinate analysis of Bray–Curtis distance with each sample colored according to the disease phenotype. PC1, PC2, and PC3 represent the top three principal coordinates that, together, captured most of the diversity. The fraction of diversity captured by the coordinate is given as a percentage. Groups were compared using PERMANOVA. d Specific bacterial-fungal correlation pattern in mice treated with vancomycin, colistin, and vehicle. Distance correlation plots of the relative abundance of fungal and bacterial families and genera. Statistical significance was determined for all pairwise comparisons; only significant correlations ( P value
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    Ro 25–6981 Mechanistic Studies Reveal UPR Modulation in Jimpy (A) qRT-PCR of Plp1 for wild-type and jimpy at day 1 of oligodendrocyte differentiation with indicated treatment. Vehicle-treated controls same as Figure S6 D. For Atf6 n = 3 technical replicates per sample. n = 4 technical replicates per sample. (B) qRT-PCR of UPR-related panel for wild-type and jimpy at day 1 of oligodendrocyte differentiation with indicated treatment. Vehicle-treated controls same as Figure S6 D. For Atf6 n = 3 technical replicates per sample. For all other probes n = 4 technical replicates per sample. Error bars represent mean ± SD. See also Figure S6 and Table S6 .

    Journal: Stem Cell Reports

    Article Title: Chemical Screening Identifies Enhancers of Mutant Oligodendrocyte Survival and Unmasks a Distinct Pathological Phase in Pelizaeus-Merzbacher Disease

    doi: 10.1016/j.stemcr.2018.07.015

    Figure Lengend Snippet: Ro 25–6981 Mechanistic Studies Reveal UPR Modulation in Jimpy (A) qRT-PCR of Plp1 for wild-type and jimpy at day 1 of oligodendrocyte differentiation with indicated treatment. Vehicle-treated controls same as Figure S6 D. For Atf6 n = 3 technical replicates per sample. n = 4 technical replicates per sample. (B) qRT-PCR of UPR-related panel for wild-type and jimpy at day 1 of oligodendrocyte differentiation with indicated treatment. Vehicle-treated controls same as Figure S6 D. For Atf6 n = 3 technical replicates per sample. For all other probes n = 4 technical replicates per sample. Error bars represent mean ± SD. See also Figure S6 and Table S6 .

    Article Snippet: qRT-PCR RNA was prepared with the RNeasy Mini Kit (74104, QIAGEN). qRT-PCR was performed using pre-designed TaqMan gene expression assays (Thermo Fisher Scientific) and probes values were normalized to an Actb endogenous control.

    Techniques: Quantitative RT-PCR

    Yy1−/−HSCs Exhibit an Aberrant Genetic Network with Corruption of Genes Involved in Cell-Cycle Regulation $$PARABREAKHERE$$RNA-seq-based comparison of gene expression in Yy1+/+ and Yy1−/−sorted HSCs. (A) Heatmap depicting statistically significant, differentially expressed upregulated and downregulated genes. (B) Gene Ontology analysis of genes upregulated in Yy1 −/− HSCs. The most enriched biological processes are shown with corresponding FDR values. F, FDR. (C) GSEA shows that genes involved in cell-cycle progression and G0 to G1 phase entry are enriched in Yy1 −/− HSCs. (D) Relative mRNA expression of genes in Lin − GFP + bone marrow cells of Yy1 +/+ versus Yy1 −/− . (E) Relative mRNA expressions of genes in Lin − GFP + bone marrow cells of Yy1 +/+ + MigR1, Yy1 −/− + YY1, or Yy1 −/− + ΔREPO. Graphs show means ± SEM; *p

    Journal: Cell reports

    Article Title: Polycomb Group Protein YY1 Is an Essential Regulator of Hematopoietic Stem Cell Quiescence

    doi: 10.1016/j.celrep.2018.01.026

    Figure Lengend Snippet: Yy1−/−HSCs Exhibit an Aberrant Genetic Network with Corruption of Genes Involved in Cell-Cycle Regulation $$PARABREAKHERE$$RNA-seq-based comparison of gene expression in Yy1+/+ and Yy1−/−sorted HSCs. (A) Heatmap depicting statistically significant, differentially expressed upregulated and downregulated genes. (B) Gene Ontology analysis of genes upregulated in Yy1 −/− HSCs. The most enriched biological processes are shown with corresponding FDR values. F, FDR. (C) GSEA shows that genes involved in cell-cycle progression and G0 to G1 phase entry are enriched in Yy1 −/− HSCs. (D) Relative mRNA expression of genes in Lin − GFP + bone marrow cells of Yy1 +/+ versus Yy1 −/− . (E) Relative mRNA expressions of genes in Lin − GFP + bone marrow cells of Yy1 +/+ + MigR1, Yy1 −/− + YY1, or Yy1 −/− + ΔREPO. Graphs show means ± SEM; *p

    Article Snippet: Total RNA was purified from bone marrow HSCs (Lin− Sca1+ c-Kit+ CD48− CD150+ and Lin− Sca1+ c-Kit+ CD48− CD150− ) sorted from Yy1 −/− versus Yy1 +/+ mice using RNeasy Micro kit (QIAGEN).

    Techniques: RNA Sequencing Assay, Expressing

    Antibiotic treatments alter fungal microbiota before and during colitis. a Fungal levels on day 0 in the fecal microbiota of mice treated with vancomycin, colistin, and vehicle were quantified using 18S rRNA qRT-PCR and were normalized to the bacterial population and quantity of DNA (μg of DNA). Data are presented as the mean ± s.e.m. b Shannon index describing the alpha diversity of the fungal microbiota (ITS2) in the fecal microbiota of mice treated with vancomycin (V), colistin (C), and vehicle (H 2 O). c Beta diversity. Principal coordinate analysis of Bray–Curtis distance with each sample colored according to the disease phenotype. PC1, PC2, and PC3 represent the top three principal coordinates that, together, captured most of the diversity. The fraction of diversity captured by the coordinate is given as a percentage. Groups were compared using PERMANOVA. d Specific bacterial-fungal correlation pattern in mice treated with vancomycin, colistin, and vehicle. Distance correlation plots of the relative abundance of fungal and bacterial families and genera. Statistical significance was determined for all pairwise comparisons; only significant correlations ( P value

    Journal: Microbiome

    Article Title: Enterobacteriaceae are essential for the modulation of colitis severity by fungi

    doi: 10.1186/s40168-018-0538-9

    Figure Lengend Snippet: Antibiotic treatments alter fungal microbiota before and during colitis. a Fungal levels on day 0 in the fecal microbiota of mice treated with vancomycin, colistin, and vehicle were quantified using 18S rRNA qRT-PCR and were normalized to the bacterial population and quantity of DNA (μg of DNA). Data are presented as the mean ± s.e.m. b Shannon index describing the alpha diversity of the fungal microbiota (ITS2) in the fecal microbiota of mice treated with vancomycin (V), colistin (C), and vehicle (H 2 O). c Beta diversity. Principal coordinate analysis of Bray–Curtis distance with each sample colored according to the disease phenotype. PC1, PC2, and PC3 represent the top three principal coordinates that, together, captured most of the diversity. The fraction of diversity captured by the coordinate is given as a percentage. Groups were compared using PERMANOVA. d Specific bacterial-fungal correlation pattern in mice treated with vancomycin, colistin, and vehicle. Distance correlation plots of the relative abundance of fungal and bacterial families and genera. Statistical significance was determined for all pairwise comparisons; only significant correlations ( P value

    Article Snippet: Gene expression analysis using quantitative reverse-transcription PCR (qRT-PCR) Total RNA was isolated from colon samples using an RNeasy Mini Kit (Qiagen, Hilden, Germany), including a DNAse treatment step, according to the manufacturer’s instructions.

    Techniques: Mouse Assay, Quantitative RT-PCR

    Enterobacteriaceae overgrowth increased fungal fitness during gut colonization. a Experimental design for the administration of colistin and vancomycin antibiotics and fungal gavages in conventional mice without DSS treatment. b Relative (left) and absolute (right) Enterobacteriaceae levels in the fecal microbiota of mice treated for 7 days with colistin, vancomycin, or vehicle (PBS) were quantified using 16S RNA qRT-PCR and were normalized to the vehicle group (PBS), day 0. Data are presented as the mean ± s.e.m. ( n = 5 per group). c C. albicans (CA) and S. boulardii (SB) counts in the feces of mice treated with colistin (C), vancomycin (V), or vehicle (PBS) and administered CA and SB by gavage. For statistical comparisons, an asterisk (*) indicates C. albicans + vancomycin versus C. albicans + colistin, and ( ) indicates S. boulardii + vancomycin vs. C. albicans + vancomycin ( n = 5 per group). d C. albicans levels (left) and S. boulardii levels (right) in the fecal microbiota were quantified using specific qRT-PCR primers and were normalized to the bacterial population ( n = 5 per group). Throughout, data are presented as the mean ± s.e.m. * P

    Journal: Microbiome

    Article Title: Enterobacteriaceae are essential for the modulation of colitis severity by fungi

    doi: 10.1186/s40168-018-0538-9

    Figure Lengend Snippet: Enterobacteriaceae overgrowth increased fungal fitness during gut colonization. a Experimental design for the administration of colistin and vancomycin antibiotics and fungal gavages in conventional mice without DSS treatment. b Relative (left) and absolute (right) Enterobacteriaceae levels in the fecal microbiota of mice treated for 7 days with colistin, vancomycin, or vehicle (PBS) were quantified using 16S RNA qRT-PCR and were normalized to the vehicle group (PBS), day 0. Data are presented as the mean ± s.e.m. ( n = 5 per group). c C. albicans (CA) and S. boulardii (SB) counts in the feces of mice treated with colistin (C), vancomycin (V), or vehicle (PBS) and administered CA and SB by gavage. For statistical comparisons, an asterisk (*) indicates C. albicans + vancomycin versus C. albicans + colistin, and ( ) indicates S. boulardii + vancomycin vs. C. albicans + vancomycin ( n = 5 per group). d C. albicans levels (left) and S. boulardii levels (right) in the fecal microbiota were quantified using specific qRT-PCR primers and were normalized to the bacterial population ( n = 5 per group). Throughout, data are presented as the mean ± s.e.m. * P

    Article Snippet: Gene expression analysis using quantitative reverse-transcription PCR (qRT-PCR) Total RNA was isolated from colon samples using an RNeasy Mini Kit (Qiagen, Hilden, Germany), including a DNAse treatment step, according to the manufacturer’s instructions.

    Techniques: Mouse Assay, Quantitative RT-PCR

    miRNA extraction substantially increases the number of miRNAs. ( A ) Comparison of array results in RNA extracted from two independent samples from the same subject. Top panel shows number of detected miRNAs. Regression analysis between the two samples is shown in the bottom panel (based on Ct values). ( B ) Comparison of array results from Qiagen RNA prep and unprocessed serum. Venn diagram of two unprocessed samples and two RNA-extracted samples from the same subject is shown in the top panel. Regression analysis between one unprocessed serum sample and one RNA-extracted sample is shown in the bottom panel (based on Ct values).

    Journal: Microrna (Shariqah, United Arab Emirates)

    Article Title: Improved Detection of Circulating miRNAs in Serum and Plasma Following Rapid Heat/Freeze Cycling

    doi: 10.2174/2211536607666180416152112

    Figure Lengend Snippet: miRNA extraction substantially increases the number of miRNAs. ( A ) Comparison of array results in RNA extracted from two independent samples from the same subject. Top panel shows number of detected miRNAs. Regression analysis between the two samples is shown in the bottom panel (based on Ct values). ( B ) Comparison of array results from Qiagen RNA prep and unprocessed serum. Venn diagram of two unprocessed samples and two RNA-extracted samples from the same subject is shown in the top panel. Regression analysis between one unprocessed serum sample and one RNA-extracted sample is shown in the bottom panel (based on Ct values).

    Article Snippet: 3.2 RNA Extraction Improves miRNA Detection To investigate whether RNA extraction/purification from serum improves miRNA detection, RNA was purified using Qiagen’s miRNeasy kit from two aliquots of serum taken from the same patient sample.

    Techniques:

    Heat/Freeze (H/F) cycle significantly improves miRNA detection. ( A ) Comparison of array results from two independent samples from the same subject after H/F process. Venn diagram between the two samples is shown in the top panel and regression analysis in the bottom panel (based on Ct values). ( B ) Comparison of array results from H/F and unprocessed serum samples. Venn diagram of two unprocessed samples and two H/F samples from the same subject is shown in the top panel. Regression analysis between one unprocessed serum sample and one H/F sample is shown in the bottom panel (based on Ct values). ( C ) Comparison of array results from a Qiagen RNA prep and Heat/Freeze (H/F) cycled serum. Top panel depicts miRNAs detected in all samples. Regression analysis between one RNA-extracted sample and one H/F processed serum sample (based on Ct values).

    Journal: Microrna (Shariqah, United Arab Emirates)

    Article Title: Improved Detection of Circulating miRNAs in Serum and Plasma Following Rapid Heat/Freeze Cycling

    doi: 10.2174/2211536607666180416152112

    Figure Lengend Snippet: Heat/Freeze (H/F) cycle significantly improves miRNA detection. ( A ) Comparison of array results from two independent samples from the same subject after H/F process. Venn diagram between the two samples is shown in the top panel and regression analysis in the bottom panel (based on Ct values). ( B ) Comparison of array results from H/F and unprocessed serum samples. Venn diagram of two unprocessed samples and two H/F samples from the same subject is shown in the top panel. Regression analysis between one unprocessed serum sample and one H/F sample is shown in the bottom panel (based on Ct values). ( C ) Comparison of array results from a Qiagen RNA prep and Heat/Freeze (H/F) cycled serum. Top panel depicts miRNAs detected in all samples. Regression analysis between one RNA-extracted sample and one H/F processed serum sample (based on Ct values).

    Article Snippet: 3.2 RNA Extraction Improves miRNA Detection To investigate whether RNA extraction/purification from serum improves miRNA detection, RNA was purified using Qiagen’s miRNeasy kit from two aliquots of serum taken from the same patient sample.

    Techniques: