quantitative pcr qpcr total rnas Search Results


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  • 99
    Thermo Fisher transcription quantitative polymerase chain reaction rt qpcr analysis total rna
    Transcription Quantitative Polymerase Chain Reaction Rt Qpcr Analysis Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 416 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore transcriptase quantitative polymerase chain reaction rt qpcr total rna
    Transcriptase Quantitative Polymerase Chain Reaction Rt Qpcr Total Rna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa quantitative pcr qpcr total rna
    Expression of miR-196a in swine tissues from 3-day-old piglets and 180-day-old adult pigs. Total <t>RNA</t> was isolated from seven different tissues including heart, liver, spleen, lung, kidney, skeletal muscle and subcutaneous adipose tissue, and the expression of miR-196a was analyzed by <t>qPCR</t> and normalized to U6.
    Quantitative Pcr Qpcr Total Rna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 96/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    FUJIFILM quantitative polymerase chain reaction qpcr total rna
    Expression of miR-196a in swine tissues from 3-day-old piglets and 180-day-old adult pigs. Total <t>RNA</t> was isolated from seven different tissues including heart, liver, spleen, lung, kidney, skeletal muscle and subcutaneous adipose tissue, and the expression of miR-196a was analyzed by <t>qPCR</t> and normalized to U6.
    Quantitative Polymerase Chain Reaction Qpcr Total Rna, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 83/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa quantitative polymerase chain reaction rt qpcr total rna
    Expression of miR-196a in swine tissues from 3-day-old piglets and 180-day-old adult pigs. Total <t>RNA</t> was isolated from seven different tissues including heart, liver, spleen, lung, kidney, skeletal muscle and subcutaneous adipose tissue, and the expression of miR-196a was analyzed by <t>qPCR</t> and normalized to U6.
    Quantitative Polymerase Chain Reaction Rt Qpcr Total Rna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 78/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa transcription quantitative polymerase chain reaction rt qpcr total rna
    siRNA for transcriptional intermediary factor 1A (TIF-IA) treatment causes Myb-binding protein 1A (MYBBP1A)-dependent p53 acetylation and apoptosis in a dose-dependent manner. ( a–e ) MCF-7 cells were transfected with the indicated concentrations of siTIF-IA. ( a ) TIF-IA mRNA levels were assessed by reverse transcription-quantitative polymerase chain reaction <t>(RT-qPCR)</t> at 60 h after transfection (n = 3). Error bars indicate mean ± standard deviation (SD). ( b ) Protein levels of TIF-IA were assessed by immunoblotting at 36 h after transfection. The middle panel is long exposure of the top panel. ( c ) pre-rRNA transcription was assessed by RT-qPCR at 60 h after transfection (n = 3). Error bars indicate mean ± SD. ( d ) Nucleolar <t>RNA</t> was isolated from purified nucleoli and quantified by spectrophotometry at 60 h after transfection (n = 3). Nucleolar RNA content of control cells was normalised to 100%. Error bars indicate mean ± SD. ( e ) Immunofluorescence staining was performed using the indicated antibodies at 60 h after transfection. ( f–h ) MCF-7 cells were transfected with combinations of siRNAs as indicated. ( f ) Cell lysates were prepared at 60 h after transfection and were immunoblotted using the indicated antibodies. Asterisk indicates nonspecific bands. ( g ) DNA content was determined by flow cytometry at 72 h after transfection (n = 3). Error bars indicate mean ± SD. ( h ) Cell lysates were prepared at 72 h after transfection and were immunoblotted using the indicated antibodies.
    Transcription Quantitative Polymerase Chain Reaction Rt Qpcr Total Rna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 112 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    82
    TaKaRa quantitative real time polymerase chain reaction qpcr total rna
    siRNA for transcriptional intermediary factor 1A (TIF-IA) treatment causes Myb-binding protein 1A (MYBBP1A)-dependent p53 acetylation and apoptosis in a dose-dependent manner. ( a–e ) MCF-7 cells were transfected with the indicated concentrations of siTIF-IA. ( a ) TIF-IA mRNA levels were assessed by reverse transcription-quantitative polymerase chain reaction <t>(RT-qPCR)</t> at 60 h after transfection (n = 3). Error bars indicate mean ± standard deviation (SD). ( b ) Protein levels of TIF-IA were assessed by immunoblotting at 36 h after transfection. The middle panel is long exposure of the top panel. ( c ) pre-rRNA transcription was assessed by RT-qPCR at 60 h after transfection (n = 3). Error bars indicate mean ± SD. ( d ) Nucleolar <t>RNA</t> was isolated from purified nucleoli and quantified by spectrophotometry at 60 h after transfection (n = 3). Nucleolar RNA content of control cells was normalised to 100%. Error bars indicate mean ± SD. ( e ) Immunofluorescence staining was performed using the indicated antibodies at 60 h after transfection. ( f–h ) MCF-7 cells were transfected with combinations of siRNAs as indicated. ( f ) Cell lysates were prepared at 60 h after transfection and were immunoblotted using the indicated antibodies. Asterisk indicates nonspecific bands. ( g ) DNA content was determined by flow cytometry at 72 h after transfection (n = 3). Error bars indicate mean ± SD. ( h ) Cell lysates were prepared at 72 h after transfection and were immunoblotted using the indicated antibodies.
    Quantitative Real Time Polymerase Chain Reaction Qpcr Total Rna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 82/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa real time quantitative pcr qpcr total rna
    Subcellular localization and encoding ability prediction of IMFNCR. (A) Intramuscular preadipocyte were stained with FITC-labeled IMFNCR probes and visualized by fluorescence. (B) The <t>RNA</t> sequences of IMFNCR, GHR-AS, and GAPDH were put into the Coding Potential Calculator (CPC) program, and both IMFNCR and GHR-AS were predicted to be non-coding RNAs, while GAPDH was identified to code for protein. (C) IMFNCR is mainly localized in the cytoplasm of intramuscular preadipocyte. RNA isolated from cytoplasm (Cyto) and nuclear (Nuc) fractions of preadipocyte and adipocyte was used to analyze the expression level of IMFNCR by semi-quantitative <t>PCR.</t> GAPDH mRNA was used as control.
    Real Time Quantitative Pcr Qpcr Total Rna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 56 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad quantitative pcr qpcr total rna
    Edited CCNI peptide level is correlated with its biological function. Correlation of CCNI peptide levels with CCNI and ADAR mRNA expression as well as Ted10 activation and Ted10 mediated tumour target killing. a Correlation between the number of edited CCNI peptide copies per cell determined by the AbsQuant ® method and mRNA editing levels determined by targeted <t>RNA-seq</t> for CCNI R75G . The scatterplot ( n = 8) includes the regression curve (red line) as well as the 95% confidence interval (grey band) and 95% prediction interval (dashed lines). b CCNI-R75G is edited by ADAR1 . HEK 293 stably expressing CCNI wildtype gene was transfected with empty vector or expression vectors of ADAR1 or ADAR2 . CCNI editing was measured by <t>RT-PCR</t> and followed by sequencing. The double peaks indicate nucleotide A to G conversion and the height of peaks reflect the level of editing. c ELISPOT assay showing IFNγ production by Ted10 incubated with peptide-pulsed or CCNI -transfected 293-A2 cells. d CTL killing assay showing that over-expression of edited CCNI gene increases the sensitivity of Ted10 mediated 293-A2 target killing ( n = 3), summarised as mean ± s.e.m. per titration. e IFNγ ELISPOT assay showing recognition of endogenous CCNI-ED antigen by Ted10. Mel-2391, mel-2400 and mel-2661 expressing both edited CCNI mRNA and HLA-A*02:01 are highly reactive to Ted10. Mel-2559, which was derived from the same patient as mel-2400 but does not have detectible edited CCNI mRNA, only reacted at background levels to Ted10. Mel-2357 and mel-2686, which express edited CCNI mRNA but do not express HLA-A*02:01, have no response to Ted10. f, Ted10 mediated target killing following incubation with mel-2400 and mel-2559 measured by caspase-3-based CTL killing assay (summarised as mean ± s.e.m. of the three triplicates)
    Quantitative Pcr Qpcr Total Rna, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 88/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad quantitative real time polymerase chain reaction qpcr total rna
    Edited CCNI peptide level is correlated with its biological function. Correlation of CCNI peptide levels with CCNI and ADAR mRNA expression as well as Ted10 activation and Ted10 mediated tumour target killing. a Correlation between the number of edited CCNI peptide copies per cell determined by the AbsQuant ® method and mRNA editing levels determined by targeted <t>RNA-seq</t> for CCNI R75G . The scatterplot ( n = 8) includes the regression curve (red line) as well as the 95% confidence interval (grey band) and 95% prediction interval (dashed lines). b CCNI-R75G is edited by ADAR1 . HEK 293 stably expressing CCNI wildtype gene was transfected with empty vector or expression vectors of ADAR1 or ADAR2 . CCNI editing was measured by <t>RT-PCR</t> and followed by sequencing. The double peaks indicate nucleotide A to G conversion and the height of peaks reflect the level of editing. c ELISPOT assay showing IFNγ production by Ted10 incubated with peptide-pulsed or CCNI -transfected 293-A2 cells. d CTL killing assay showing that over-expression of edited CCNI gene increases the sensitivity of Ted10 mediated 293-A2 target killing ( n = 3), summarised as mean ± s.e.m. per titration. e IFNγ ELISPOT assay showing recognition of endogenous CCNI-ED antigen by Ted10. Mel-2391, mel-2400 and mel-2661 expressing both edited CCNI mRNA and HLA-A*02:01 are highly reactive to Ted10. Mel-2559, which was derived from the same patient as mel-2400 but does not have detectible edited CCNI mRNA, only reacted at background levels to Ted10. Mel-2357 and mel-2686, which express edited CCNI mRNA but do not express HLA-A*02:01, have no response to Ted10. f, Ted10 mediated target killing following incubation with mel-2400 and mel-2559 measured by caspase-3-based CTL killing assay (summarised as mean ± s.e.m. of the three triplicates)
    Quantitative Real Time Polymerase Chain Reaction Qpcr Total Rna, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher quantitative reverse transcription polymerase chain reaction qpcr total rna
    Edited CCNI peptide level is correlated with its biological function. Correlation of CCNI peptide levels with CCNI and ADAR mRNA expression as well as Ted10 activation and Ted10 mediated tumour target killing. a Correlation between the number of edited CCNI peptide copies per cell determined by the AbsQuant ® method and mRNA editing levels determined by targeted <t>RNA-seq</t> for CCNI R75G . The scatterplot ( n = 8) includes the regression curve (red line) as well as the 95% confidence interval (grey band) and 95% prediction interval (dashed lines). b CCNI-R75G is edited by ADAR1 . HEK 293 stably expressing CCNI wildtype gene was transfected with empty vector or expression vectors of ADAR1 or ADAR2 . CCNI editing was measured by <t>RT-PCR</t> and followed by sequencing. The double peaks indicate nucleotide A to G conversion and the height of peaks reflect the level of editing. c ELISPOT assay showing IFNγ production by Ted10 incubated with peptide-pulsed or CCNI -transfected 293-A2 cells. d CTL killing assay showing that over-expression of edited CCNI gene increases the sensitivity of Ted10 mediated 293-A2 target killing ( n = 3), summarised as mean ± s.e.m. per titration. e IFNγ ELISPOT assay showing recognition of endogenous CCNI-ED antigen by Ted10. Mel-2391, mel-2400 and mel-2661 expressing both edited CCNI mRNA and HLA-A*02:01 are highly reactive to Ted10. Mel-2559, which was derived from the same patient as mel-2400 but does not have detectible edited CCNI mRNA, only reacted at background levels to Ted10. Mel-2357 and mel-2686, which express edited CCNI mRNA but do not express HLA-A*02:01, have no response to Ted10. f, Ted10 mediated target killing following incubation with mel-2400 and mel-2559 measured by caspase-3-based CTL killing assay (summarised as mean ± s.e.m. of the three triplicates)
    Quantitative Reverse Transcription Polymerase Chain Reaction Qpcr Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Stratagene quantitative pcr qpcr total rna
    Knockdown of endogenous nuclear IL-33 expression in primary human endothelial cells using two independent <t>RNA</t> silencing strategies. ( a,b ) Nuclear expression of endogenous IL-33 in confluent monolayers of primary human endothelial cells. IL-33 protein (red) was detected by indirect immunofluorescence staining with anti-IL-33 mAbs Nessy1 ( a ) and 305B ( b ). Nuclei were counterstained with DAPI (blue). ( c ) Knockdown of endogenous nuclear IL-33 expression. IL-33 expression was analyzed by indirect immunofluorescence with Nessy1 mAb 48 h after transfection of a pool of IL-33 siRNAs (siIL-33-Sm ) or control siRNA ( siCTL-Sm ). ( d–g ) Validation of the two independent RNA silencing strategies. Expression of IL-33 was analyzed at the protein level by western blot ( d,f ) and RNA level by <t>qPCR</t> ( e,g ), 72 h after the second siRNA transfection. Cells were treated with distinct pools of IL-33 siRNAs , siIL-33-Sm ( d,e ) or siIL-33-Mi ( f,g ), and corresponding controls, siCTL-Sm ( d,e ) and siCTL-Mi ( f,g ). RRL IL-33, human IL-33 protein produced by in vitro translation in rabbit reticulocyte lysates ( d,f ). For the qPCR experiments, relative mRNA levels were calculated by normalizing the signals to those of actin. Results are shown as means with s.d. from three separate datapoints. ( h,i ). Knockdown of endogenous nuclear IL-33 does not affect NFkB protein expression. Normalized protein quantities deduced from all peptides intensity values (LFQ intensities) are shown for NFkB p65 (RELA), NFkB p105 (NFKB1) and NFkB p100 (NFKB2). Endothelial cells were treated with siIL-33-Sm ( h ) or siIL-33-Mi ( i ), and corresponding controls. Results are shown as means with s.d. from three biological replicate experiments.
    Quantitative Pcr Qpcr Total Rna, supplied by Stratagene, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa qpcr human reference total rna
    Knockdown of endogenous nuclear IL-33 expression in primary human endothelial cells using two independent <t>RNA</t> silencing strategies. ( a,b ) Nuclear expression of endogenous IL-33 in confluent monolayers of primary human endothelial cells. IL-33 protein (red) was detected by indirect immunofluorescence staining with anti-IL-33 mAbs Nessy1 ( a ) and 305B ( b ). Nuclei were counterstained with DAPI (blue). ( c ) Knockdown of endogenous nuclear IL-33 expression. IL-33 expression was analyzed by indirect immunofluorescence with Nessy1 mAb 48 h after transfection of a pool of IL-33 siRNAs (siIL-33-Sm ) or control siRNA ( siCTL-Sm ). ( d–g ) Validation of the two independent RNA silencing strategies. Expression of IL-33 was analyzed at the protein level by western blot ( d,f ) and RNA level by <t>qPCR</t> ( e,g ), 72 h after the second siRNA transfection. Cells were treated with distinct pools of IL-33 siRNAs , siIL-33-Sm ( d,e ) or siIL-33-Mi ( f,g ), and corresponding controls, siCTL-Sm ( d,e ) and siCTL-Mi ( f,g ). RRL IL-33, human IL-33 protein produced by in vitro translation in rabbit reticulocyte lysates ( d,f ). For the qPCR experiments, relative mRNA levels were calculated by normalizing the signals to those of actin. Results are shown as means with s.d. from three separate datapoints. ( h,i ). Knockdown of endogenous nuclear IL-33 does not affect NFkB protein expression. Normalized protein quantities deduced from all peptides intensity values (LFQ intensities) are shown for NFkB p65 (RELA), NFkB p105 (NFKB1) and NFkB p100 (NFKB2). Endothelial cells were treated with siIL-33-Sm ( h ) or siIL-33-Mi ( i ), and corresponding controls. Results are shown as means with s.d. from three biological replicate experiments.
    Qpcr Human Reference Total Rna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 59 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies quantitative pcr qpcr total rna
    miR-135a-5p downregulates NHE9 protein expression. a Raw expression values of miR-135a, miR-124 and miR-153 were obtained from human miRNA tissue atlas. MiRNA abundance in brain from tissue biopsies of two normal (i.e. cancer free) individuals was conducted using SurePrint 8 × 60 K Human V19 and V21 miRNA microarray analysis as previously described [ 30 ]. b Comparative analysis of NHE9 mRNA and miR-135a expression profiles across various tissues. NHE9 mRNA expression profile was obtained from <t>RNA-seq</t> analysis performed of human tissue samples from 95 normal (i.e. cancer free) individuals as described previously [ 31 ]. MiRNA expression profile was obtained from human miRNA tissue atlas [ 30 ]. Normalization was done as a percentage relative to tissue with highest expression. c Mir-135a expression levels in U87 and U251n cell lines relative to normal human brain tissue. d <t>qPCR</t> analysis of miR-135a from U87 cells transfected with miR-135a mimic relative to control U87 cells. e Immunoblots of U87 cell lysates transfected with miR-135a or scrambled control mimic were probed using anti-NHE9 and anti-tubulin antibodies. f NHE9 protein expression levels determined by western blotting. Graphs represent average band intensity from densitometric scans of immunoblots from three biological replicates. Error bars represent standard deviation (SD), * p
    Quantitative Pcr Qpcr Total Rna, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 86/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Omega Bio-tek reverse transcription quantitative polymerase chain reaction rt qpcr total rna kit ii kit
    miR-135a-5p downregulates NHE9 protein expression. a Raw expression values of miR-135a, miR-124 and miR-153 were obtained from human miRNA tissue atlas. MiRNA abundance in brain from tissue biopsies of two normal (i.e. cancer free) individuals was conducted using SurePrint 8 × 60 K Human V19 and V21 miRNA microarray analysis as previously described [ 30 ]. b Comparative analysis of NHE9 mRNA and miR-135a expression profiles across various tissues. NHE9 mRNA expression profile was obtained from <t>RNA-seq</t> analysis performed of human tissue samples from 95 normal (i.e. cancer free) individuals as described previously [ 31 ]. MiRNA expression profile was obtained from human miRNA tissue atlas [ 30 ]. Normalization was done as a percentage relative to tissue with highest expression. c Mir-135a expression levels in U87 and U251n cell lines relative to normal human brain tissue. d <t>qPCR</t> analysis of miR-135a from U87 cells transfected with miR-135a mimic relative to control U87 cells. e Immunoblots of U87 cell lysates transfected with miR-135a or scrambled control mimic were probed using anti-NHE9 and anti-tubulin antibodies. f NHE9 protein expression levels determined by western blotting. Graphs represent average band intensity from densitometric scans of immunoblots from three biological replicates. Error bars represent standard deviation (SD), * p
    Reverse Transcription Quantitative Polymerase Chain Reaction Rt Qpcr Total Rna Kit Ii Kit, supplied by Omega Bio-tek, used in various techniques. Bioz Stars score: 89/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad qpcr total reference rna
    miR-135a-5p downregulates NHE9 protein expression. a Raw expression values of miR-135a, miR-124 and miR-153 were obtained from human miRNA tissue atlas. MiRNA abundance in brain from tissue biopsies of two normal (i.e. cancer free) individuals was conducted using SurePrint 8 × 60 K Human V19 and V21 miRNA microarray analysis as previously described [ 30 ]. b Comparative analysis of NHE9 mRNA and miR-135a expression profiles across various tissues. NHE9 mRNA expression profile was obtained from <t>RNA-seq</t> analysis performed of human tissue samples from 95 normal (i.e. cancer free) individuals as described previously [ 31 ]. MiRNA expression profile was obtained from human miRNA tissue atlas [ 30 ]. Normalization was done as a percentage relative to tissue with highest expression. c Mir-135a expression levels in U87 and U251n cell lines relative to normal human brain tissue. d <t>qPCR</t> analysis of miR-135a from U87 cells transfected with miR-135a mimic relative to control U87 cells. e Immunoblots of U87 cell lysates transfected with miR-135a or scrambled control mimic were probed using anti-NHE9 and anti-tubulin antibodies. f NHE9 protein expression levels determined by western blotting. Graphs represent average band intensity from densitometric scans of immunoblots from three biological replicates. Error bars represent standard deviation (SD), * p
    Qpcr Total Reference Rna, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 79/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa quantitative polymerase chain reaction pcr total rna
    miR-135a-5p downregulates NHE9 protein expression. a Raw expression values of miR-135a, miR-124 and miR-153 were obtained from human miRNA tissue atlas. MiRNA abundance in brain from tissue biopsies of two normal (i.e. cancer free) individuals was conducted using SurePrint 8 × 60 K Human V19 and V21 miRNA microarray analysis as previously described [ 30 ]. b Comparative analysis of NHE9 mRNA and miR-135a expression profiles across various tissues. NHE9 mRNA expression profile was obtained from <t>RNA-seq</t> analysis performed of human tissue samples from 95 normal (i.e. cancer free) individuals as described previously [ 31 ]. MiRNA expression profile was obtained from human miRNA tissue atlas [ 30 ]. Normalization was done as a percentage relative to tissue with highest expression. c Mir-135a expression levels in U87 and U251n cell lines relative to normal human brain tissue. d <t>qPCR</t> analysis of miR-135a from U87 cells transfected with miR-135a mimic relative to control U87 cells. e Immunoblots of U87 cell lysates transfected with miR-135a or scrambled control mimic were probed using anti-NHE9 and anti-tubulin antibodies. f NHE9 protein expression levels determined by western blotting. Graphs represent average band intensity from densitometric scans of immunoblots from three biological replicates. Error bars represent standard deviation (SD), * p
    Quantitative Polymerase Chain Reaction Pcr Total Rna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 79/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa quantitative polymerase chain reaction
    miR-135a-5p downregulates NHE9 protein expression. a Raw expression values of miR-135a, miR-124 and miR-153 were obtained from human miRNA tissue atlas. MiRNA abundance in brain from tissue biopsies of two normal (i.e. cancer free) individuals was conducted using SurePrint 8 × 60 K Human V19 and V21 miRNA microarray analysis as previously described [ 30 ]. b Comparative analysis of NHE9 mRNA and miR-135a expression profiles across various tissues. NHE9 mRNA expression profile was obtained from <t>RNA-seq</t> analysis performed of human tissue samples from 95 normal (i.e. cancer free) individuals as described previously [ 31 ]. MiRNA expression profile was obtained from human miRNA tissue atlas [ 30 ]. Normalization was done as a percentage relative to tissue with highest expression. c Mir-135a expression levels in U87 and U251n cell lines relative to normal human brain tissue. d <t>qPCR</t> analysis of miR-135a from U87 cells transfected with miR-135a mimic relative to control U87 cells. e Immunoblots of U87 cell lysates transfected with miR-135a or scrambled control mimic were probed using anti-NHE9 and anti-tubulin antibodies. f NHE9 protein expression levels determined by western blotting. Graphs represent average band intensity from densitometric scans of immunoblots from three biological replicates. Error bars represent standard deviation (SD), * p
    Quantitative Polymerase Chain Reaction, supplied by TaKaRa, used in various techniques. Bioz Stars score: 91/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher quantitative polymerase chain reaction rt qpcr total normal brain rna
    miR-331-3p is down regulated in GBM cell lines compared to normal brain and inhibits cell proliferation and clonogenicity. a TaqMan <t>RT-qPCR</t> analysis of miR-331-3p expression in U-87 MG and U-251 MG cells relative to normal brain <t>RNA.</t> b Cell titre assay of U-251 MG cell proliferation following transient transfection with either miR-331-3p or negative control miRNA (miR-NC). c Clonogenicity assay of U-251 MG cells 12 days following transient transfection with either miR-331-3p or miR-NC. Error bars represent standard deviations; * p
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    Roche quantitative polymerase chain reaction pcr total rna
    MOV10 contributes to the regulation of INK4a in primary fibroblasts. ( a ) FDF cells were infected with lentiviruses encoding a control shRNA (Ctrl) and two independent shRNAs against MOV10 and CBX7 respectively (sh1 and sh2). The knockdown efficiency and the effect on p16 INK4a were assessed by immunoblotting with antibodies against MOV10, CBX7 and p16 INK4a . GAPDH was used as a loading control. ( b ) Effects of MOV10 and CBX7 shRNAs on INK4a and ARF <t>RNA</t> levels as determined by <t>qRT-PCR.</t> Error bars, s.d.; n =3. ( c ) Phase contrast photographs showing the enlarged and flattened appearance of cells expressing the MOV10 and CBX7 shRNAs. ( d ) Following knockdown of MOV10 (as in panel a ), cell lysates were immunoblotted for p53 and p21 CIP1 as indicated. GAPDH was used as a loading control.
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    Molecular Research Center inc quantitative polymerase chain reaction pcr total rna
    Comparing the expression of glucocorticoid-regulated genes in inguinal fat of CRH-Tg and wild type mice. Total <t>RNA</t> of inguinal fat of wild type and CRH-Tg mice were isolated and converted to cDNA. Real-time <t>PCR</t> was then performed to monitor the expression of genes indicated. Data shows fold-induction of gene expression (CRH-Tg/wild type) from 6–8 mice (*, p
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    Thermo Fisher reverse transcription quantitative polymerase chain reaction rt pcr trizol
    Comparing the expression of glucocorticoid-regulated genes in inguinal fat of CRH-Tg and wild type mice. Total <t>RNA</t> of inguinal fat of wild type and CRH-Tg mice were isolated and converted to cDNA. Real-time <t>PCR</t> was then performed to monitor the expression of genes indicated. Data shows fold-induction of gene expression (CRH-Tg/wild type) from 6–8 mice (*, p
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    TaKaRa absolute qpcr human total rnas
    Comparing the expression of glucocorticoid-regulated genes in inguinal fat of CRH-Tg and wild type mice. Total <t>RNA</t> of inguinal fat of wild type and CRH-Tg mice were isolated and converted to cDNA. Real-time <t>PCR</t> was then performed to monitor the expression of genes indicated. Data shows fold-induction of gene expression (CRH-Tg/wild type) from 6–8 mice (*, p
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    Stratagene quantitative real time pcr qpcr total rna
    Nuclear translocation of Trx1 in response to oscillatory shear stress and stimulated NF-κB activation and proinflammatory gene expression by increased nuclear Trx1. EC exposed either LS or OS for 24 h were fractionated to obtain cytosolic and nuclear fractions. A. Cytosolic (left) and nuclear (right) fractions were examined for Trx1 expression level by Western blotting probed with an antibody specific to Trx1. To verify each compartment, cytosolic and nuclear fractions were probed with β-actin and lamin antibodies, respectively. B. EC transfected with NLS-Trx1 or VC were exposed to LS or OS for 24 h and nuclear fractions were examined for NF-κB activity by EMSA. NLS-Trx1 expression measured by Western blotting with antibody specific to Myc epitope is shown on right, top. C. Total <t>RNA</t> was isolated from EC exposed to LS or OS after NLS-Trx1 transfection. cDNA obtained from reverse transcription of total RNA were quantitated for ICAM1 and IL-6 genes by qRT- <t>PCR.</t> Data are mean ± SE (* p
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    Stratagene qpcr human reference total rna
    Nuclear translocation of Trx1 in response to oscillatory shear stress and stimulated NF-κB activation and proinflammatory gene expression by increased nuclear Trx1. EC exposed either LS or OS for 24 h were fractionated to obtain cytosolic and nuclear fractions. A. Cytosolic (left) and nuclear (right) fractions were examined for Trx1 expression level by Western blotting probed with an antibody specific to Trx1. To verify each compartment, cytosolic and nuclear fractions were probed with β-actin and lamin antibodies, respectively. B. EC transfected with NLS-Trx1 or VC were exposed to LS or OS for 24 h and nuclear fractions were examined for NF-κB activity by EMSA. NLS-Trx1 expression measured by Western blotting with antibody specific to Myc epitope is shown on right, top. C. Total <t>RNA</t> was isolated from EC exposed to LS or OS after NLS-Trx1 transfection. cDNA obtained from reverse transcription of total RNA were quantitated for ICAM1 and IL-6 genes by qRT- <t>PCR.</t> Data are mean ± SE (* p
    Qpcr Human Reference Total Rna, supplied by Stratagene, used in various techniques. Bioz Stars score: 98/100, based on 109 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    OriGene quantitative polymerase chain reaction q pcr total rna
    Nuclear translocation of Trx1 in response to oscillatory shear stress and stimulated NF-κB activation and proinflammatory gene expression by increased nuclear Trx1. EC exposed either LS or OS for 24 h were fractionated to obtain cytosolic and nuclear fractions. A. Cytosolic (left) and nuclear (right) fractions were examined for Trx1 expression level by Western blotting probed with an antibody specific to Trx1. To verify each compartment, cytosolic and nuclear fractions were probed with β-actin and lamin antibodies, respectively. B. EC transfected with NLS-Trx1 or VC were exposed to LS or OS for 24 h and nuclear fractions were examined for NF-κB activity by EMSA. NLS-Trx1 expression measured by Western blotting with antibody specific to Myc epitope is shown on right, top. C. Total <t>RNA</t> was isolated from EC exposed to LS or OS after NLS-Trx1 transfection. cDNA obtained from reverse transcription of total RNA were quantitated for ICAM1 and IL-6 genes by qRT- <t>PCR.</t> Data are mean ± SE (* p
    Quantitative Polymerase Chain Reaction Q Pcr Total Rna, supplied by OriGene, used in various techniques. Bioz Stars score: 79/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Omega Bio-tek quantitative pcr total rna
    RNAi-mediated knockdown of ERI3 and infection with DENV2, YFV17D or EV71. HuH-7 or RD cells were transfected using 15 nM of the indicated siRNA and incubated for 48 hours. For DENV-2 and YFV17D infections, transfected HuH-7 cells were infected with an MOI = 1.0 and incubated 24 hours. For EV71 infection, transfected RD cells were infected with an MOI = 0.3 and incubated 24 hours. Cells and tissue culture supernatants were harvested and assayed for protein by western blot, <t>RNA</t> by quantitative real time <t>PCR</t> and infectious particle production by focus forming assay (DENV2 and YFV17D) or plaque assay (EV71). ( A,D , G) Western blotting to test for knockdown of ERI3. Blots were probed with antibodies specific to actin or ERI3 and visualized using the Licor Odyssey Imaging system. ( B,E , H ) Quantitative real time PCR on total cellular RNA to assay for viral RNA content. Random cDNA was generated from total RNA and used in quantitative real time PCR using primers specific for GAPDH and DENV-2, YFV17D or EV71. Fold-change (∆∆Ct) relative to the control knockdown was calculated using GAPDH and viral RNA Ct values. Statistical significance was determined using a one-way ANOVA (CI = 95%) comparing samples to the siRNA control samples. ( C,F , I ) Focus or plaque assays for infectious particle production. Tissue culture supernatants from DENV-2 or YFV17D infections were used to inoculate BHK cell monolayers followed by overlay with CMC. Titer was determined by probing fixed cell monolayers using the pan-flaviviral Env antibody, 4G2 and anti-mouse DyLight680 secondary antibody and visualized using the Licor Odyssey Imaging system. Titer is expressed as focus forming units (FFU)/mL. Tissue culture supernatants from EV71 infection was used to inoculate Vero cell monolayers followed by overlay with CMC. Titer was determined by fixing the cell monolayers and staining with 1% Crystal Violet solution. Titer is expressed as plaque forming units (PFU)/mL. Statistical significance was determined using a one-way ANOVA (CI = 95%) comparing samples to the siRNA control samples. All knockdowns and infections were performed in triplicate in three independent experiments.
    Quantitative Pcr Total Rna, supplied by Omega Bio-tek, used in various techniques. Bioz Stars score: 97/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Meridian Life Science qpcr total rna
    RNAi-mediated knockdown of ERI3 and infection with DENV2, YFV17D or EV71. HuH-7 or RD cells were transfected using 15 nM of the indicated siRNA and incubated for 48 hours. For DENV-2 and YFV17D infections, transfected HuH-7 cells were infected with an MOI = 1.0 and incubated 24 hours. For EV71 infection, transfected RD cells were infected with an MOI = 0.3 and incubated 24 hours. Cells and tissue culture supernatants were harvested and assayed for protein by western blot, <t>RNA</t> by quantitative real time <t>PCR</t> and infectious particle production by focus forming assay (DENV2 and YFV17D) or plaque assay (EV71). ( A,D , G) Western blotting to test for knockdown of ERI3. Blots were probed with antibodies specific to actin or ERI3 and visualized using the Licor Odyssey Imaging system. ( B,E , H ) Quantitative real time PCR on total cellular RNA to assay for viral RNA content. Random cDNA was generated from total RNA and used in quantitative real time PCR using primers specific for GAPDH and DENV-2, YFV17D or EV71. Fold-change (∆∆Ct) relative to the control knockdown was calculated using GAPDH and viral RNA Ct values. Statistical significance was determined using a one-way ANOVA (CI = 95%) comparing samples to the siRNA control samples. ( C,F , I ) Focus or plaque assays for infectious particle production. Tissue culture supernatants from DENV-2 or YFV17D infections were used to inoculate BHK cell monolayers followed by overlay with CMC. Titer was determined by probing fixed cell monolayers using the pan-flaviviral Env antibody, 4G2 and anti-mouse DyLight680 secondary antibody and visualized using the Licor Odyssey Imaging system. Titer is expressed as focus forming units (FFU)/mL. Tissue culture supernatants from EV71 infection was used to inoculate Vero cell monolayers followed by overlay with CMC. Titer was determined by fixing the cell monolayers and staining with 1% Crystal Violet solution. Titer is expressed as plaque forming units (PFU)/mL. Statistical significance was determined using a one-way ANOVA (CI = 95%) comparing samples to the siRNA control samples. All knockdowns and infections were performed in triplicate in three independent experiments.
    Qpcr Total Rna, supplied by Meridian Life Science, used in various techniques. Bioz Stars score: 98/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher mirna quantitative polymerase chain reaction qpcr validation total rna
    Differential <t>miRNA</t> expression underwent (A) <t>qPCR</t> validation and (B) target genes of the differentially expressed miRNAs were detected by qPCR. Data are presented as the means ± standard deviation, n=8. ** P
    Mirna Quantitative Polymerase Chain Reaction Qpcr Validation Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 568 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies qpcr human reference total rna
    Differential <t>miRNA</t> expression underwent (A) <t>qPCR</t> validation and (B) target genes of the differentially expressed miRNAs were detected by qPCR. Data are presented as the means ± standard deviation, n=8. ** P
    Qpcr Human Reference Total Rna, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 92/100, based on 54 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Expression of miR-196a in swine tissues from 3-day-old piglets and 180-day-old adult pigs. Total RNA was isolated from seven different tissues including heart, liver, spleen, lung, kidney, skeletal muscle and subcutaneous adipose tissue, and the expression of miR-196a was analyzed by qPCR and normalized to U6.

    Journal: Genes

    Article Title: Expression Profiles and Biological Roles of miR-196a in Swine

    doi: 10.3390/genes7020005

    Figure Lengend Snippet: Expression of miR-196a in swine tissues from 3-day-old piglets and 180-day-old adult pigs. Total RNA was isolated from seven different tissues including heart, liver, spleen, lung, kidney, skeletal muscle and subcutaneous adipose tissue, and the expression of miR-196a was analyzed by qPCR and normalized to U6.

    Article Snippet: 2.9. mRNA and miRNA Quantification by qPCR Total RNA was prepared from frozen tissue or cells using TRIzol reagent (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer’s protocol.

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

    Isolation and cultivation of DPCs and BMSCs from GFP-transgenic rats, and expression of spinal cord injury repair-associated cytokines. (A-G) Isolation and cultivation of DPCs and BMSCs from GFP-transgenic rats. (A) Direct GFP-fluorescence and (B) phase-contrast images demonstrated the morphological characteristics of primary DPCs. (C and D) DPCs at passage 6. (E) Direct GFP-fluorescence and (F) phase-contrast images revealed the morphological characteristics of BMSCs. (G and H) BMSCs at passage 6. (I) Semi-quantitative RT-PCR analysis was performed to confirm the expression of NTFs, angiogenic factors and inflammation-associated cytokines in DPCs. (J) RT-qPCR analysis further revealed relative expression levels of NTFs, angiogenic factors and inflammation-associated cytokines in DPCs. *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Rat vibrissa dermal papilla cells promote healing of spinal cord injury following transplantation

    doi: 10.3892/etm.2018.5916

    Figure Lengend Snippet: Isolation and cultivation of DPCs and BMSCs from GFP-transgenic rats, and expression of spinal cord injury repair-associated cytokines. (A-G) Isolation and cultivation of DPCs and BMSCs from GFP-transgenic rats. (A) Direct GFP-fluorescence and (B) phase-contrast images demonstrated the morphological characteristics of primary DPCs. (C and D) DPCs at passage 6. (E) Direct GFP-fluorescence and (F) phase-contrast images revealed the morphological characteristics of BMSCs. (G and H) BMSCs at passage 6. (I) Semi-quantitative RT-PCR analysis was performed to confirm the expression of NTFs, angiogenic factors and inflammation-associated cytokines in DPCs. (J) RT-qPCR analysis further revealed relative expression levels of NTFs, angiogenic factors and inflammation-associated cytokines in DPCs. *P

    Article Snippet: RT-quantitative PCR (RT-qPCR) Total RNA from DPCs and BMSCs was extracted using TRIzol reagent, according to the manufacturer's protocols, then reverse transcribed into cDNA using the Takara RNA PCR kit (AMV) Version 3.0 (Takara Biotechnology Co., Ltd.) according to the manufacturer's protocols.

    Techniques: Isolation, Transgenic Assay, Expressing, Fluorescence, Quantitative RT-PCR

    siRNA for transcriptional intermediary factor 1A (TIF-IA) treatment causes Myb-binding protein 1A (MYBBP1A)-dependent p53 acetylation and apoptosis in a dose-dependent manner. ( a–e ) MCF-7 cells were transfected with the indicated concentrations of siTIF-IA. ( a ) TIF-IA mRNA levels were assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) at 60 h after transfection (n = 3). Error bars indicate mean ± standard deviation (SD). ( b ) Protein levels of TIF-IA were assessed by immunoblotting at 36 h after transfection. The middle panel is long exposure of the top panel. ( c ) pre-rRNA transcription was assessed by RT-qPCR at 60 h after transfection (n = 3). Error bars indicate mean ± SD. ( d ) Nucleolar RNA was isolated from purified nucleoli and quantified by spectrophotometry at 60 h after transfection (n = 3). Nucleolar RNA content of control cells was normalised to 100%. Error bars indicate mean ± SD. ( e ) Immunofluorescence staining was performed using the indicated antibodies at 60 h after transfection. ( f–h ) MCF-7 cells were transfected with combinations of siRNAs as indicated. ( f ) Cell lysates were prepared at 60 h after transfection and were immunoblotted using the indicated antibodies. Asterisk indicates nonspecific bands. ( g ) DNA content was determined by flow cytometry at 72 h after transfection (n = 3). Error bars indicate mean ± SD. ( h ) Cell lysates were prepared at 72 h after transfection and were immunoblotted using the indicated antibodies.

    Journal: Scientific Reports

    Article Title: Gradual reduction in rRNA transcription triggers p53 acetylation and apoptosis via MYBBP1A

    doi: 10.1038/srep10854

    Figure Lengend Snippet: siRNA for transcriptional intermediary factor 1A (TIF-IA) treatment causes Myb-binding protein 1A (MYBBP1A)-dependent p53 acetylation and apoptosis in a dose-dependent manner. ( a–e ) MCF-7 cells were transfected with the indicated concentrations of siTIF-IA. ( a ) TIF-IA mRNA levels were assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) at 60 h after transfection (n = 3). Error bars indicate mean ± standard deviation (SD). ( b ) Protein levels of TIF-IA were assessed by immunoblotting at 36 h after transfection. The middle panel is long exposure of the top panel. ( c ) pre-rRNA transcription was assessed by RT-qPCR at 60 h after transfection (n = 3). Error bars indicate mean ± SD. ( d ) Nucleolar RNA was isolated from purified nucleoli and quantified by spectrophotometry at 60 h after transfection (n = 3). Nucleolar RNA content of control cells was normalised to 100%. Error bars indicate mean ± SD. ( e ) Immunofluorescence staining was performed using the indicated antibodies at 60 h after transfection. ( f–h ) MCF-7 cells were transfected with combinations of siRNAs as indicated. ( f ) Cell lysates were prepared at 60 h after transfection and were immunoblotted using the indicated antibodies. Asterisk indicates nonspecific bands. ( g ) DNA content was determined by flow cytometry at 72 h after transfection (n = 3). Error bars indicate mean ± SD. ( h ) Cell lysates were prepared at 72 h after transfection and were immunoblotted using the indicated antibodies.

    Article Snippet: RNA purification and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) Total RNA was isolated with a FastPure® RNA kit (Takara Bio, Shiga, Japan) according to the manufacturer’s instructions.

    Techniques: IA, Binding Assay, Transfection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Standard Deviation, Isolation, Purification, Spectrophotometry, Immunofluorescence, Staining, Flow Cytometry, Cytometry

    Depleting rRNA processing factors causes RPL11-dependent and Myb-binding protein 1A (MYBBP1A)-independent p53 activation. ( a–c ) MCF-7 cells were transfected with siCtrl, siPES1, siWDR3, siNOL1 or siUTP6. ( a ) Expression of rRNA processing factors was assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) at 48 h after transfection (n = 3). Error bars indicate mean ± SD. ( b ) Protein levels of rRNA processing factors were assessed by immunoblotting at 48 h after transfection. ( c ) Northern blotting was performed at 60 h after transfection using a probe specific for the rRNA region. An ethidium bromide-stained gel is shown at the bottom as the loading control. ( d–g ) MCF-7 cells were transfected with combinations of siRNAs as indicated. ( d ) Cell lysates were prepared at 60 h after transfection and immunoblotted using the indicated antibodies. Lysates from 2 μg/ml camptothecin (CPT)-treated cells were used as the positive control. ( e ) DNA content was determined by flow cytometry at 72 h after transfection (n = 3). Error bars indicate mean ± standard deviation (SD). ( f ) Cell lysates were prepared at 72 h after transfection and immunoblotted using the indicated antibodies. ( g ) Cell lysates were prepared at 60 h after transfection and immunoblotted using the indicated antibodies. Lysates from 2 μg/ml CPT-treated cells were used as the positive control. ( h ) Nucleolar RNA content was spectrophotometrically quantified at 60 h after transfection (n = 3). Nucleolar RNA content of control cells was normalised to 100%. Error bars indicate mean ± SD. (i) Immunofluorescence staining was performed using the indicated antibodies at 60 h after transfection.

    Journal: Scientific Reports

    Article Title: Gradual reduction in rRNA transcription triggers p53 acetylation and apoptosis via MYBBP1A

    doi: 10.1038/srep10854

    Figure Lengend Snippet: Depleting rRNA processing factors causes RPL11-dependent and Myb-binding protein 1A (MYBBP1A)-independent p53 activation. ( a–c ) MCF-7 cells were transfected with siCtrl, siPES1, siWDR3, siNOL1 or siUTP6. ( a ) Expression of rRNA processing factors was assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) at 48 h after transfection (n = 3). Error bars indicate mean ± SD. ( b ) Protein levels of rRNA processing factors were assessed by immunoblotting at 48 h after transfection. ( c ) Northern blotting was performed at 60 h after transfection using a probe specific for the rRNA region. An ethidium bromide-stained gel is shown at the bottom as the loading control. ( d–g ) MCF-7 cells were transfected with combinations of siRNAs as indicated. ( d ) Cell lysates were prepared at 60 h after transfection and immunoblotted using the indicated antibodies. Lysates from 2 μg/ml camptothecin (CPT)-treated cells were used as the positive control. ( e ) DNA content was determined by flow cytometry at 72 h after transfection (n = 3). Error bars indicate mean ± standard deviation (SD). ( f ) Cell lysates were prepared at 72 h after transfection and immunoblotted using the indicated antibodies. ( g ) Cell lysates were prepared at 60 h after transfection and immunoblotted using the indicated antibodies. Lysates from 2 μg/ml CPT-treated cells were used as the positive control. ( h ) Nucleolar RNA content was spectrophotometrically quantified at 60 h after transfection (n = 3). Nucleolar RNA content of control cells was normalised to 100%. Error bars indicate mean ± SD. (i) Immunofluorescence staining was performed using the indicated antibodies at 60 h after transfection.

    Article Snippet: RNA purification and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) Total RNA was isolated with a FastPure® RNA kit (Takara Bio, Shiga, Japan) according to the manufacturer’s instructions.

    Techniques: Binding Assay, Activation Assay, Transfection, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Northern Blot, Staining, Cycling Probe Technology, Positive Control, Flow Cytometry, Cytometry, Standard Deviation, Immunofluorescence

    Subcellular localization and encoding ability prediction of IMFNCR. (A) Intramuscular preadipocyte were stained with FITC-labeled IMFNCR probes and visualized by fluorescence. (B) The RNA sequences of IMFNCR, GHR-AS, and GAPDH were put into the Coding Potential Calculator (CPC) program, and both IMFNCR and GHR-AS were predicted to be non-coding RNAs, while GAPDH was identified to code for protein. (C) IMFNCR is mainly localized in the cytoplasm of intramuscular preadipocyte. RNA isolated from cytoplasm (Cyto) and nuclear (Nuc) fractions of preadipocyte and adipocyte was used to analyze the expression level of IMFNCR by semi-quantitative PCR. GAPDH mRNA was used as control.

    Journal: Frontiers in Genetics

    Article Title: LncRNA IMFNCR Promotes Intramuscular Adipocyte Differentiation by Sponging miR-128-3p and miR-27b-3p

    doi: 10.3389/fgene.2019.00042

    Figure Lengend Snippet: Subcellular localization and encoding ability prediction of IMFNCR. (A) Intramuscular preadipocyte were stained with FITC-labeled IMFNCR probes and visualized by fluorescence. (B) The RNA sequences of IMFNCR, GHR-AS, and GAPDH were put into the Coding Potential Calculator (CPC) program, and both IMFNCR and GHR-AS were predicted to be non-coding RNAs, while GAPDH was identified to code for protein. (C) IMFNCR is mainly localized in the cytoplasm of intramuscular preadipocyte. RNA isolated from cytoplasm (Cyto) and nuclear (Nuc) fractions of preadipocyte and adipocyte was used to analyze the expression level of IMFNCR by semi-quantitative PCR. GAPDH mRNA was used as control.

    Article Snippet: RNA Isolation and Real-Time Quantitative PCR (qPCR) Total RNA from tissues and preadipocyte were isolated using extracted with Trizol reagent according to the manufacturer’s protocol (Takara, Dalian, China).

    Techniques: Staining, Labeling, Fluorescence, Isolation, Expressing, Real-time Polymerase Chain Reaction

    Characteristics of chicken IMFNCR in different tissues and cells. (A) Tissue expression profile of IMFNCR by qRT-PCR. (B) Tissue expression profile of IMFNCR by semi-quantitative PCR. (C) The expression dynamics of IMFNCR in breast muscle tissues at different physiological periods. (D) The expression level of IMFNCR in abdominal and intramuscular preadipocyte. Data represent means ± SEM ( n = 3). ∗ p ≤ 0.05; ∗∗ p ≤ 0.01.

    Journal: Frontiers in Genetics

    Article Title: LncRNA IMFNCR Promotes Intramuscular Adipocyte Differentiation by Sponging miR-128-3p and miR-27b-3p

    doi: 10.3389/fgene.2019.00042

    Figure Lengend Snippet: Characteristics of chicken IMFNCR in different tissues and cells. (A) Tissue expression profile of IMFNCR by qRT-PCR. (B) Tissue expression profile of IMFNCR by semi-quantitative PCR. (C) The expression dynamics of IMFNCR in breast muscle tissues at different physiological periods. (D) The expression level of IMFNCR in abdominal and intramuscular preadipocyte. Data represent means ± SEM ( n = 3). ∗ p ≤ 0.05; ∗∗ p ≤ 0.01.

    Article Snippet: RNA Isolation and Real-Time Quantitative PCR (qPCR) Total RNA from tissues and preadipocyte were isolated using extracted with Trizol reagent according to the manufacturer’s protocol (Takara, Dalian, China).

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

    IMFNCR promotes intramuscular adipocyte differentiation by sponging miR-128-3p and miR-27b-3p. (A) The putative miR-128-3p and miR-27b-3p-binding sites at PPARG 3′UTR (green) are evolutionarily conserved across species. (B) miR-128-3p and miR-27b-3p suppresses PPARG translation. miR-128-3p and miR-27b-3p were transfected into DF1 cells, along with PPARG-UTR-WT but not PPARG-UTR-Mut. The luciferase activity was analyzed 48 h later. (C) miR-128-3p suppresses the expression of PPARG and IRS1. Intramuscular preadipocyte was infected with miR-128-3p mimics or mimics Control at 37°C, followed by the addition of fresh growth medium. RNA and protein were extracted 48 h later, and levels were accessed by qRT-PCR and (D) western blot. (E) IMFNCR siRNA and inhibitor (miR-128-3p inhibitor, mixture inhibitor) or mimics (miR-128-3p mimics, mixture mimics) were transfected into chicken intramuscular preadipocyte. qRT-PCR assays were performed to determine the expression levels of PPARG and IMFNCR. (F) Negative control inhibitor (NC inhibitor) or miR-128-3p or mixture (miR-128-3p and miR-27b-3p) inhibitor was transfected into chicken intramuscular preadipocyte. Forty-eight hours later, qRT-PCR assays were performed to determine the expression levels of miR-128-3p, miR-27b-3p, PPARG, and FABP4. (G) IMFNCR siRNA and miR-27b-3p were transfected into miRNA mimics NC or miR-128-3p mimics. At 48 h intracellular triglyceride content was measured by Oil Red O staining and (H) quantified by microplate reader at 510 nm. The data represent means ± SEM ( n = 3). ∗ p ≤ 0.05; ∗∗ p ≤ 0.01.

    Journal: Frontiers in Genetics

    Article Title: LncRNA IMFNCR Promotes Intramuscular Adipocyte Differentiation by Sponging miR-128-3p and miR-27b-3p

    doi: 10.3389/fgene.2019.00042

    Figure Lengend Snippet: IMFNCR promotes intramuscular adipocyte differentiation by sponging miR-128-3p and miR-27b-3p. (A) The putative miR-128-3p and miR-27b-3p-binding sites at PPARG 3′UTR (green) are evolutionarily conserved across species. (B) miR-128-3p and miR-27b-3p suppresses PPARG translation. miR-128-3p and miR-27b-3p were transfected into DF1 cells, along with PPARG-UTR-WT but not PPARG-UTR-Mut. The luciferase activity was analyzed 48 h later. (C) miR-128-3p suppresses the expression of PPARG and IRS1. Intramuscular preadipocyte was infected with miR-128-3p mimics or mimics Control at 37°C, followed by the addition of fresh growth medium. RNA and protein were extracted 48 h later, and levels were accessed by qRT-PCR and (D) western blot. (E) IMFNCR siRNA and inhibitor (miR-128-3p inhibitor, mixture inhibitor) or mimics (miR-128-3p mimics, mixture mimics) were transfected into chicken intramuscular preadipocyte. qRT-PCR assays were performed to determine the expression levels of PPARG and IMFNCR. (F) Negative control inhibitor (NC inhibitor) or miR-128-3p or mixture (miR-128-3p and miR-27b-3p) inhibitor was transfected into chicken intramuscular preadipocyte. Forty-eight hours later, qRT-PCR assays were performed to determine the expression levels of miR-128-3p, miR-27b-3p, PPARG, and FABP4. (G) IMFNCR siRNA and miR-27b-3p were transfected into miRNA mimics NC or miR-128-3p mimics. At 48 h intracellular triglyceride content was measured by Oil Red O staining and (H) quantified by microplate reader at 510 nm. The data represent means ± SEM ( n = 3). ∗ p ≤ 0.05; ∗∗ p ≤ 0.01.

    Article Snippet: RNA Isolation and Real-Time Quantitative PCR (qPCR) Total RNA from tissues and preadipocyte were isolated using extracted with Trizol reagent according to the manufacturer’s protocol (Takara, Dalian, China).

    Techniques: Binding Assay, Transfection, Luciferase, Activity Assay, Expressing, Infection, Quantitative RT-PCR, Western Blot, Negative Control, Staining

    DDR1 expression is increased in PDAC at mRNA level. a increased DDR1 mRNA expression in 30 matched tumor (T) and non-tumor tissue (N) was detected by real-time quantitative PCR. b DDR1 expression in Buchholz pancreas grouped by normal pancreatic duct (1) and PDAC (2)

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: High expression of DDR1 is associated with the poor prognosis in Chinese patients with pancreatic ductal adenocarcinoma

    doi: 10.1186/s13046-015-0202-1

    Figure Lengend Snippet: DDR1 expression is increased in PDAC at mRNA level. a increased DDR1 mRNA expression in 30 matched tumor (T) and non-tumor tissue (N) was detected by real-time quantitative PCR. b DDR1 expression in Buchholz pancreas grouped by normal pancreatic duct (1) and PDAC (2)

    Article Snippet: RNA extraction and real-time quantitative PCR (RT-qPCR) Total RNA from primary tumor and adjacent non-tumor tissue samples was isolated with Trizol reagent (Takara, Japan), and reversely transcribed through PrimeScript RT-qPCR kit (Takara, Japan) according to the manufacturer’s instructions [ ].

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Edited CCNI peptide level is correlated with its biological function. Correlation of CCNI peptide levels with CCNI and ADAR mRNA expression as well as Ted10 activation and Ted10 mediated tumour target killing. a Correlation between the number of edited CCNI peptide copies per cell determined by the AbsQuant ® method and mRNA editing levels determined by targeted RNA-seq for CCNI R75G . The scatterplot ( n = 8) includes the regression curve (red line) as well as the 95% confidence interval (grey band) and 95% prediction interval (dashed lines). b CCNI-R75G is edited by ADAR1 . HEK 293 stably expressing CCNI wildtype gene was transfected with empty vector or expression vectors of ADAR1 or ADAR2 . CCNI editing was measured by RT-PCR and followed by sequencing. The double peaks indicate nucleotide A to G conversion and the height of peaks reflect the level of editing. c ELISPOT assay showing IFNγ production by Ted10 incubated with peptide-pulsed or CCNI -transfected 293-A2 cells. d CTL killing assay showing that over-expression of edited CCNI gene increases the sensitivity of Ted10 mediated 293-A2 target killing ( n = 3), summarised as mean ± s.e.m. per titration. e IFNγ ELISPOT assay showing recognition of endogenous CCNI-ED antigen by Ted10. Mel-2391, mel-2400 and mel-2661 expressing both edited CCNI mRNA and HLA-A*02:01 are highly reactive to Ted10. Mel-2559, which was derived from the same patient as mel-2400 but does not have detectible edited CCNI mRNA, only reacted at background levels to Ted10. Mel-2357 and mel-2686, which express edited CCNI mRNA but do not express HLA-A*02:01, have no response to Ted10. f, Ted10 mediated target killing following incubation with mel-2400 and mel-2559 measured by caspase-3-based CTL killing assay (summarised as mean ± s.e.m. of the three triplicates)

    Journal: Nature Communications

    Article Title: RNA editing derived epitopes function as cancer antigens to elicit immune responses

    doi: 10.1038/s41467-018-06405-9

    Figure Lengend Snippet: Edited CCNI peptide level is correlated with its biological function. Correlation of CCNI peptide levels with CCNI and ADAR mRNA expression as well as Ted10 activation and Ted10 mediated tumour target killing. a Correlation between the number of edited CCNI peptide copies per cell determined by the AbsQuant ® method and mRNA editing levels determined by targeted RNA-seq for CCNI R75G . The scatterplot ( n = 8) includes the regression curve (red line) as well as the 95% confidence interval (grey band) and 95% prediction interval (dashed lines). b CCNI-R75G is edited by ADAR1 . HEK 293 stably expressing CCNI wildtype gene was transfected with empty vector or expression vectors of ADAR1 or ADAR2 . CCNI editing was measured by RT-PCR and followed by sequencing. The double peaks indicate nucleotide A to G conversion and the height of peaks reflect the level of editing. c ELISPOT assay showing IFNγ production by Ted10 incubated with peptide-pulsed or CCNI -transfected 293-A2 cells. d CTL killing assay showing that over-expression of edited CCNI gene increases the sensitivity of Ted10 mediated 293-A2 target killing ( n = 3), summarised as mean ± s.e.m. per titration. e IFNγ ELISPOT assay showing recognition of endogenous CCNI-ED antigen by Ted10. Mel-2391, mel-2400 and mel-2661 expressing both edited CCNI mRNA and HLA-A*02:01 are highly reactive to Ted10. Mel-2559, which was derived from the same patient as mel-2400 but does not have detectible edited CCNI mRNA, only reacted at background levels to Ted10. Mel-2357 and mel-2686, which express edited CCNI mRNA but do not express HLA-A*02:01, have no response to Ted10. f, Ted10 mediated target killing following incubation with mel-2400 and mel-2559 measured by caspase-3-based CTL killing assay (summarised as mean ± s.e.m. of the three triplicates)

    Article Snippet: Quantitative PCR (qPCR) Total RNA was isolated and converted to cDNA as described above. qPCR was performed using iTaq™ Universal SYBR® Green Supermix reagent (Bio Red, 1725122) in a C1000TM Thermal Cycler CFX96 Real-Time System following manufacturer’s instructions (Bio-Rad).

    Techniques: Expressing, Activation Assay, RNA Sequencing Assay, Stable Transfection, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Sequencing, Enzyme-linked Immunospot, Incubation, CTL Assay, Over Expression, Titration, Derivative Assay

    Knockdown of endogenous nuclear IL-33 expression in primary human endothelial cells using two independent RNA silencing strategies. ( a,b ) Nuclear expression of endogenous IL-33 in confluent monolayers of primary human endothelial cells. IL-33 protein (red) was detected by indirect immunofluorescence staining with anti-IL-33 mAbs Nessy1 ( a ) and 305B ( b ). Nuclei were counterstained with DAPI (blue). ( c ) Knockdown of endogenous nuclear IL-33 expression. IL-33 expression was analyzed by indirect immunofluorescence with Nessy1 mAb 48 h after transfection of a pool of IL-33 siRNAs (siIL-33-Sm ) or control siRNA ( siCTL-Sm ). ( d–g ) Validation of the two independent RNA silencing strategies. Expression of IL-33 was analyzed at the protein level by western blot ( d,f ) and RNA level by qPCR ( e,g ), 72 h after the second siRNA transfection. Cells were treated with distinct pools of IL-33 siRNAs , siIL-33-Sm ( d,e ) or siIL-33-Mi ( f,g ), and corresponding controls, siCTL-Sm ( d,e ) and siCTL-Mi ( f,g ). RRL IL-33, human IL-33 protein produced by in vitro translation in rabbit reticulocyte lysates ( d,f ). For the qPCR experiments, relative mRNA levels were calculated by normalizing the signals to those of actin. Results are shown as means with s.d. from three separate datapoints. ( h,i ). Knockdown of endogenous nuclear IL-33 does not affect NFkB protein expression. Normalized protein quantities deduced from all peptides intensity values (LFQ intensities) are shown for NFkB p65 (RELA), NFkB p105 (NFKB1) and NFkB p100 (NFKB2). Endothelial cells were treated with siIL-33-Sm ( h ) or siIL-33-Mi ( i ), and corresponding controls. Results are shown as means with s.d. from three biological replicate experiments.

    Journal: Scientific Reports

    Article Title: Extracellular IL-33 cytokine, but not endogenous nuclear IL-33, regulates protein expression in endothelial cells

    doi: 10.1038/srep34255

    Figure Lengend Snippet: Knockdown of endogenous nuclear IL-33 expression in primary human endothelial cells using two independent RNA silencing strategies. ( a,b ) Nuclear expression of endogenous IL-33 in confluent monolayers of primary human endothelial cells. IL-33 protein (red) was detected by indirect immunofluorescence staining with anti-IL-33 mAbs Nessy1 ( a ) and 305B ( b ). Nuclei were counterstained with DAPI (blue). ( c ) Knockdown of endogenous nuclear IL-33 expression. IL-33 expression was analyzed by indirect immunofluorescence with Nessy1 mAb 48 h after transfection of a pool of IL-33 siRNAs (siIL-33-Sm ) or control siRNA ( siCTL-Sm ). ( d–g ) Validation of the two independent RNA silencing strategies. Expression of IL-33 was analyzed at the protein level by western blot ( d,f ) and RNA level by qPCR ( e,g ), 72 h after the second siRNA transfection. Cells were treated with distinct pools of IL-33 siRNAs , siIL-33-Sm ( d,e ) or siIL-33-Mi ( f,g ), and corresponding controls, siCTL-Sm ( d,e ) and siCTL-Mi ( f,g ). RRL IL-33, human IL-33 protein produced by in vitro translation in rabbit reticulocyte lysates ( d,f ). For the qPCR experiments, relative mRNA levels were calculated by normalizing the signals to those of actin. Results are shown as means with s.d. from three separate datapoints. ( h,i ). Knockdown of endogenous nuclear IL-33 does not affect NFkB protein expression. Normalized protein quantities deduced from all peptides intensity values (LFQ intensities) are shown for NFkB p65 (RELA), NFkB p105 (NFKB1) and NFkB p100 (NFKB2). Endothelial cells were treated with siIL-33-Sm ( h ) or siIL-33-Mi ( i ), and corresponding controls. Results are shown as means with s.d. from three biological replicate experiments.

    Article Snippet: Quantitative PCR (qPCR) Total RNA was isolated using the Absolute RNA Kit from Stratagene (Agilent Technologies) and cDNAs were synthesized using SuperSript III First strand cDNA synthesis system for RT-PCR (Invitrogen) according to manufacturer’s instructions. qPCR was performed using the ABI7500 Prism SDS Real-Time PCR Detection System (Applied Biosystems) with a SYBR Green PCR Master Mix kit (Applied Biosystems) and a standard temperature protocol.

    Techniques: Expressing, Immunofluorescence, Staining, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Produced, In Vitro

    miR-135a-5p downregulates NHE9 protein expression. a Raw expression values of miR-135a, miR-124 and miR-153 were obtained from human miRNA tissue atlas. MiRNA abundance in brain from tissue biopsies of two normal (i.e. cancer free) individuals was conducted using SurePrint 8 × 60 K Human V19 and V21 miRNA microarray analysis as previously described [ 30 ]. b Comparative analysis of NHE9 mRNA and miR-135a expression profiles across various tissues. NHE9 mRNA expression profile was obtained from RNA-seq analysis performed of human tissue samples from 95 normal (i.e. cancer free) individuals as described previously [ 31 ]. MiRNA expression profile was obtained from human miRNA tissue atlas [ 30 ]. Normalization was done as a percentage relative to tissue with highest expression. c Mir-135a expression levels in U87 and U251n cell lines relative to normal human brain tissue. d qPCR analysis of miR-135a from U87 cells transfected with miR-135a mimic relative to control U87 cells. e Immunoblots of U87 cell lysates transfected with miR-135a or scrambled control mimic were probed using anti-NHE9 and anti-tubulin antibodies. f NHE9 protein expression levels determined by western blotting. Graphs represent average band intensity from densitometric scans of immunoblots from three biological replicates. Error bars represent standard deviation (SD), * p

    Journal: Cell Communication and Signaling : CCS

    Article Title: MicroRNA-135a regulates NHE9 to inhibit proliferation and migration of glioblastoma cells

    doi: 10.1186/s12964-017-0209-7

    Figure Lengend Snippet: miR-135a-5p downregulates NHE9 protein expression. a Raw expression values of miR-135a, miR-124 and miR-153 were obtained from human miRNA tissue atlas. MiRNA abundance in brain from tissue biopsies of two normal (i.e. cancer free) individuals was conducted using SurePrint 8 × 60 K Human V19 and V21 miRNA microarray analysis as previously described [ 30 ]. b Comparative analysis of NHE9 mRNA and miR-135a expression profiles across various tissues. NHE9 mRNA expression profile was obtained from RNA-seq analysis performed of human tissue samples from 95 normal (i.e. cancer free) individuals as described previously [ 31 ]. MiRNA expression profile was obtained from human miRNA tissue atlas [ 30 ]. Normalization was done as a percentage relative to tissue with highest expression. c Mir-135a expression levels in U87 and U251n cell lines relative to normal human brain tissue. d qPCR analysis of miR-135a from U87 cells transfected with miR-135a mimic relative to control U87 cells. e Immunoblots of U87 cell lysates transfected with miR-135a or scrambled control mimic were probed using anti-NHE9 and anti-tubulin antibodies. f NHE9 protein expression levels determined by western blotting. Graphs represent average band intensity from densitometric scans of immunoblots from three biological replicates. Error bars represent standard deviation (SD), * p

    Article Snippet: RNA isolation and qPCR Total RNA from human brain tissue was obtained from Agilent Technologies.

    Techniques: Expressing, Microarray, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Transfection, Western Blot, Standard Deviation

    miR-331-3p is down regulated in GBM cell lines compared to normal brain and inhibits cell proliferation and clonogenicity. a TaqMan RT-qPCR analysis of miR-331-3p expression in U-87 MG and U-251 MG cells relative to normal brain RNA. b Cell titre assay of U-251 MG cell proliferation following transient transfection with either miR-331-3p or negative control miRNA (miR-NC). c Clonogenicity assay of U-251 MG cells 12 days following transient transfection with either miR-331-3p or miR-NC. Error bars represent standard deviations; * p

    Journal: Journal of Neuro-Oncology

    Article Title: miR-331-3p regulates expression of neuropilin-2 in glioblastoma

    doi: 10.1007/s11060-013-1271-7

    Figure Lengend Snippet: miR-331-3p is down regulated in GBM cell lines compared to normal brain and inhibits cell proliferation and clonogenicity. a TaqMan RT-qPCR analysis of miR-331-3p expression in U-87 MG and U-251 MG cells relative to normal brain RNA. b Cell titre assay of U-251 MG cell proliferation following transient transfection with either miR-331-3p or negative control miRNA (miR-NC). c Clonogenicity assay of U-251 MG cells 12 days following transient transfection with either miR-331-3p or miR-NC. Error bars represent standard deviations; * p

    Article Snippet: RNA extraction, reverse transcription and quantitative polymerase chain reaction (RT-qPCR) Total normal brain RNA was obtained from Ambion (Cat #AM7962) and used as a reference control sample.

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

    MOV10 contributes to the regulation of INK4a in primary fibroblasts. ( a ) FDF cells were infected with lentiviruses encoding a control shRNA (Ctrl) and two independent shRNAs against MOV10 and CBX7 respectively (sh1 and sh2). The knockdown efficiency and the effect on p16 INK4a were assessed by immunoblotting with antibodies against MOV10, CBX7 and p16 INK4a . GAPDH was used as a loading control. ( b ) Effects of MOV10 and CBX7 shRNAs on INK4a and ARF RNA levels as determined by qRT-PCR. Error bars, s.d.; n =3. ( c ) Phase contrast photographs showing the enlarged and flattened appearance of cells expressing the MOV10 and CBX7 shRNAs. ( d ) Following knockdown of MOV10 (as in panel a ), cell lysates were immunoblotted for p53 and p21 CIP1 as indicated. GAPDH was used as a loading control.

    Journal: Nature structural & molecular biology

    Article Title: Role for the MOV10 RNA helicase in Polycomb-mediated repression of the INK4a tumor suppressor

    doi: 10.1038/nsmb.1824

    Figure Lengend Snippet: MOV10 contributes to the regulation of INK4a in primary fibroblasts. ( a ) FDF cells were infected with lentiviruses encoding a control shRNA (Ctrl) and two independent shRNAs against MOV10 and CBX7 respectively (sh1 and sh2). The knockdown efficiency and the effect on p16 INK4a were assessed by immunoblotting with antibodies against MOV10, CBX7 and p16 INK4a . GAPDH was used as a loading control. ( b ) Effects of MOV10 and CBX7 shRNAs on INK4a and ARF RNA levels as determined by qRT-PCR. Error bars, s.d.; n =3. ( c ) Phase contrast photographs showing the enlarged and flattened appearance of cells expressing the MOV10 and CBX7 shRNAs. ( d ) Following knockdown of MOV10 (as in panel a ), cell lysates were immunoblotted for p53 and p21 CIP1 as indicated. GAPDH was used as a loading control.

    Article Snippet: RNA extraction, quantitative reverse transcription and quantitative PCR Total RNA was prepared using the Ultra Pure RNA extraction Kit from Roche and the cDNA was generated using 0.25–1 μg of RNA using MultiScribe reverse transcriptase and random hexamer primers (Applied Biosystems).

    Techniques: Infection, shRNA, Quantitative RT-PCR, Expressing

    Comparing the expression of glucocorticoid-regulated genes in inguinal fat of CRH-Tg and wild type mice. Total RNA of inguinal fat of wild type and CRH-Tg mice were isolated and converted to cDNA. Real-time PCR was then performed to monitor the expression of genes indicated. Data shows fold-induction of gene expression (CRH-Tg/wild type) from 6–8 mice (*, p

    Journal: PLoS ONE

    Article Title: Genome-Wide Analysis of Glucocorticoid Receptor Binding Regions in Adipocytes Reveal Gene Network Involved in Triglyceride Homeostasis

    doi: 10.1371/journal.pone.0015188

    Figure Lengend Snippet: Comparing the expression of glucocorticoid-regulated genes in inguinal fat of CRH-Tg and wild type mice. Total RNA of inguinal fat of wild type and CRH-Tg mice were isolated and converted to cDNA. Real-time PCR was then performed to monitor the expression of genes indicated. Data shows fold-induction of gene expression (CRH-Tg/wild type) from 6–8 mice (*, p

    Article Snippet: RNA isolation and quantitative PCR Total RNA was isolated from mouse inguinal fat using TRI Reagent® RT (Molecular Research Center, Inc.).

    Techniques: Expressing, Mouse Assay, Isolation, Real-time Polymerase Chain Reaction

    Nuclear translocation of Trx1 in response to oscillatory shear stress and stimulated NF-κB activation and proinflammatory gene expression by increased nuclear Trx1. EC exposed either LS or OS for 24 h were fractionated to obtain cytosolic and nuclear fractions. A. Cytosolic (left) and nuclear (right) fractions were examined for Trx1 expression level by Western blotting probed with an antibody specific to Trx1. To verify each compartment, cytosolic and nuclear fractions were probed with β-actin and lamin antibodies, respectively. B. EC transfected with NLS-Trx1 or VC were exposed to LS or OS for 24 h and nuclear fractions were examined for NF-κB activity by EMSA. NLS-Trx1 expression measured by Western blotting with antibody specific to Myc epitope is shown on right, top. C. Total RNA was isolated from EC exposed to LS or OS after NLS-Trx1 transfection. cDNA obtained from reverse transcription of total RNA were quantitated for ICAM1 and IL-6 genes by qRT- PCR. Data are mean ± SE (* p

    Journal: PLoS ONE

    Article Title: Disturbed Flow Enhances Inflammatory Signaling and Atherogenesis by Increasing Thioredoxin-1 Level in Endothelial Cell Nuclei

    doi: 10.1371/journal.pone.0108346

    Figure Lengend Snippet: Nuclear translocation of Trx1 in response to oscillatory shear stress and stimulated NF-κB activation and proinflammatory gene expression by increased nuclear Trx1. EC exposed either LS or OS for 24 h were fractionated to obtain cytosolic and nuclear fractions. A. Cytosolic (left) and nuclear (right) fractions were examined for Trx1 expression level by Western blotting probed with an antibody specific to Trx1. To verify each compartment, cytosolic and nuclear fractions were probed with β-actin and lamin antibodies, respectively. B. EC transfected with NLS-Trx1 or VC were exposed to LS or OS for 24 h and nuclear fractions were examined for NF-κB activity by EMSA. NLS-Trx1 expression measured by Western blotting with antibody specific to Myc epitope is shown on right, top. C. Total RNA was isolated from EC exposed to LS or OS after NLS-Trx1 transfection. cDNA obtained from reverse transcription of total RNA were quantitated for ICAM1 and IL-6 genes by qRT- PCR. Data are mean ± SE (* p

    Article Snippet: Quantitative real-time PCR (qPCR) Total RNA was polyadenylated and reverse transcribed for use in a two-step qRT-PCR using the High-capacity cDNA Synthesis kit (ABI) and qRT-PCR kits (Stratagene) as described .

    Techniques: Translocation Assay, Activation Assay, Expressing, Western Blot, Transfection, Activity Assay, Isolation, Quantitative RT-PCR

    RNAi-mediated knockdown of ERI3 and infection with DENV2, YFV17D or EV71. HuH-7 or RD cells were transfected using 15 nM of the indicated siRNA and incubated for 48 hours. For DENV-2 and YFV17D infections, transfected HuH-7 cells were infected with an MOI = 1.0 and incubated 24 hours. For EV71 infection, transfected RD cells were infected with an MOI = 0.3 and incubated 24 hours. Cells and tissue culture supernatants were harvested and assayed for protein by western blot, RNA by quantitative real time PCR and infectious particle production by focus forming assay (DENV2 and YFV17D) or plaque assay (EV71). ( A,D , G) Western blotting to test for knockdown of ERI3. Blots were probed with antibodies specific to actin or ERI3 and visualized using the Licor Odyssey Imaging system. ( B,E , H ) Quantitative real time PCR on total cellular RNA to assay for viral RNA content. Random cDNA was generated from total RNA and used in quantitative real time PCR using primers specific for GAPDH and DENV-2, YFV17D or EV71. Fold-change (∆∆Ct) relative to the control knockdown was calculated using GAPDH and viral RNA Ct values. Statistical significance was determined using a one-way ANOVA (CI = 95%) comparing samples to the siRNA control samples. ( C,F , I ) Focus or plaque assays for infectious particle production. Tissue culture supernatants from DENV-2 or YFV17D infections were used to inoculate BHK cell monolayers followed by overlay with CMC. Titer was determined by probing fixed cell monolayers using the pan-flaviviral Env antibody, 4G2 and anti-mouse DyLight680 secondary antibody and visualized using the Licor Odyssey Imaging system. Titer is expressed as focus forming units (FFU)/mL. Tissue culture supernatants from EV71 infection was used to inoculate Vero cell monolayers followed by overlay with CMC. Titer was determined by fixing the cell monolayers and staining with 1% Crystal Violet solution. Titer is expressed as plaque forming units (PFU)/mL. Statistical significance was determined using a one-way ANOVA (CI = 95%) comparing samples to the siRNA control samples. All knockdowns and infections were performed in triplicate in three independent experiments.

    Journal: Scientific Reports

    Article Title: The Golgi associated ERI3 is a Flavivirus host factor

    doi: 10.1038/srep34379

    Figure Lengend Snippet: RNAi-mediated knockdown of ERI3 and infection with DENV2, YFV17D or EV71. HuH-7 or RD cells were transfected using 15 nM of the indicated siRNA and incubated for 48 hours. For DENV-2 and YFV17D infections, transfected HuH-7 cells were infected with an MOI = 1.0 and incubated 24 hours. For EV71 infection, transfected RD cells were infected with an MOI = 0.3 and incubated 24 hours. Cells and tissue culture supernatants were harvested and assayed for protein by western blot, RNA by quantitative real time PCR and infectious particle production by focus forming assay (DENV2 and YFV17D) or plaque assay (EV71). ( A,D , G) Western blotting to test for knockdown of ERI3. Blots were probed with antibodies specific to actin or ERI3 and visualized using the Licor Odyssey Imaging system. ( B,E , H ) Quantitative real time PCR on total cellular RNA to assay for viral RNA content. Random cDNA was generated from total RNA and used in quantitative real time PCR using primers specific for GAPDH and DENV-2, YFV17D or EV71. Fold-change (∆∆Ct) relative to the control knockdown was calculated using GAPDH and viral RNA Ct values. Statistical significance was determined using a one-way ANOVA (CI = 95%) comparing samples to the siRNA control samples. ( C,F , I ) Focus or plaque assays for infectious particle production. Tissue culture supernatants from DENV-2 or YFV17D infections were used to inoculate BHK cell monolayers followed by overlay with CMC. Titer was determined by probing fixed cell monolayers using the pan-flaviviral Env antibody, 4G2 and anti-mouse DyLight680 secondary antibody and visualized using the Licor Odyssey Imaging system. Titer is expressed as focus forming units (FFU)/mL. Tissue culture supernatants from EV71 infection was used to inoculate Vero cell monolayers followed by overlay with CMC. Titer was determined by fixing the cell monolayers and staining with 1% Crystal Violet solution. Titer is expressed as plaque forming units (PFU)/mL. Statistical significance was determined using a one-way ANOVA (CI = 95%) comparing samples to the siRNA control samples. All knockdowns and infections were performed in triplicate in three independent experiments.

    Article Snippet: Quantitative PCR Total RNA was extracted from cells using the EZNA Total RNA kit (Omega Bio-Tek) and cDNA was synthesized using iScript (BioRad) according to the manufacturers’ instructions.

    Techniques: Infection, Transfection, Incubation, Western Blot, Real-time Polymerase Chain Reaction, Focus Forming Assay, Plaque Assay, Imaging, Generated, Staining

    Differential miRNA expression underwent (A) qPCR validation and (B) target genes of the differentially expressed miRNAs were detected by qPCR. Data are presented as the means ± standard deviation, n=8. ** P

    Journal: International Journal of Molecular Medicine

    Article Title: Maternal chromium restriction induces insulin resistance in adult mice offspring through miRNA

    doi: 10.3892/ijmm.2017.3328

    Figure Lengend Snippet: Differential miRNA expression underwent (A) qPCR validation and (B) target genes of the differentially expressed miRNAs were detected by qPCR. Data are presented as the means ± standard deviation, n=8. ** P

    Article Snippet: miRNA quantitative polymerase chain reaction (qPCR) validation Total RNA obained using the mirVana™ RNA Isolation kit mentioned above was quantified using a NanoDrop 1000, and then reverse transcribed using TaqMan MicroRNA RT kit and RT primers from the respective TaqMan MicroRNA assay kit. qPCR was performed on an ABI 7900 thermocycler using the TaqMan Universal PCR Master Mix and TaqMan probes from the TaqMan MicroRNA assay kit (all from Applied Biosystems; Thermo Fisher Scientific, Inc.).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Standard Deviation