real-time pcr total rna Search Results


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  • 99
    Thermo Fisher real time pcr total rna
    M6P/IGF2R expression increases during monocyte differentiation to macrophages . ( A ) Cell‐surface expression of M6P/IGF2R on isolated PBMC and MACS‐enriched monocytes from healthy donors was evaluated with mAb MEM‐238‐AF647 and flow cytometry. In parallel, MOPC‐21‐AF647 was used as an isotype control mAb, displayed by the cut‐off gates. The same analysis was performed with macrophages differentiated from MACS‐sorted monocytes during a 7‐day culture with recombinant human M‐CSF (50 ng/ml) followed by 2 days resting in macrophage serum free medium. ( B ) Primary human monocytes and monocyte‐derived macrophages from (A) were lysed and <t>RNA</t> was extracted. cDNA was synthesized from total RNA and gene expression was measured by real‐time <t>PCR</t> as described in the Material and Methods section with TaqMan primer sets for human M6P/IGF2R and YWHAZ as endogenous control. The M6P/IGF2R mean expression values relative to that of monocytes ± SD from 3 donors is shown
    Real Time Pcr Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 16008 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore real time pcr analysis total rna
    HS-induced transcription of Hsp70, Hsp83 and Hsp22 genes is decreased in Chd1 -deficient and Chd1 C D -rescued larvae. Larvae bearing the indicated transgenes in a Chd1 2 /Exel7014 genetic background and wild-type larvae were subjected to the indicated periods of HS before <t>RNA</t> was prepared and expression of the HS genes was analyzed by real-time <t>PCR.</t> All values were normalized against the non-HS (0) sample of the respective fly strain. Error bars indicate standard deviations of three independent experiments. ( A ) Hsp70 , ( B ) Hsp83 and ( C ) Hsp22 .
    Real Time Pcr Analysis Total Rna, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 194 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher quantitative real time pcr assay total rna
    HMMR-AS1 stabilizes HMMR mRNA. (A) Relative levels of HMMR-AS1 and HMMR mRNA in U87 and HA cells transfected with lentivirus containing HMMR-AS1 full-length sequence compare with control. (B) HMMR protein expression tested by Western blot in cells that HMMR-AS1 was downregulated or upregulated. (C) HMMR protein expression tested by immunofluorescence in control cells or cells with HMMR-AS1 or HMMR overexpression. Scale bar = 10 μm. (D) U87 cells differently transfected were treated with 50 μM α-amanitin for 6, 12, or 18 hours. Cells were then harvested for <t>qRT-PCR</t> to test the loss of HMMR mRNA. 18s <t>RNA,</t> a product of RNA polymerase I, was not affected by α-amanitin. Cells transfected with HMMR shRNA were used as positive control. β-Actin was used as a loading control for Western blot. The blot bands were quantified by ImageJ and represented by relative values compare with loading control (1.00). # P > .05, * P
    Quantitative Real Time Pcr Assay Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 247 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher quantitative real time pcr analysis total rna
    Quantitative <t>RT-PCR</t> analysis of the expression of β-LCY1 , β-LCY2 , and β-CHX genes in the flavedo and the pulp of Navel orange ( C. sinensis ) (black bars) and Star Ruby grapefruit ( C. paradisi ) (white bars) at the IG (immature green), MG (mature green), B (breaker), and FC (full-colour) stages. The levels of expression were normalized to the amount of <t>RNA</t> and the value of Navel flavedo at the FC stage was set to 100. The data are means ±SD of three experimental replicates.
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    Millipore real time pcr total rna
    Quantitative <t>RT-PCR</t> analysis of the expression of β-LCY1 , β-LCY2 , and β-CHX genes in the flavedo and the pulp of Navel orange ( C. sinensis ) (black bars) and Star Ruby grapefruit ( C. paradisi ) (white bars) at the IG (immature green), MG (mature green), B (breaker), and FC (full-colour) stages. The levels of expression were normalized to the amount of <t>RNA</t> and the value of Navel flavedo at the FC stage was set to 100. The data are means ±SD of three experimental replicates.
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    TaKaRa quantitative real time pcr total rnas
    NDUFA4L2 is involved in the survival and migration of non‐small cell lung cancer (NSCLC) in hypoxia. ( a ) NDUFA4L2 messenger <t>RNA</t> (mRNA) of human NSCLC tissue samples was measured by quantitative <t>PCR.</t> ( b ) Four pairs of tissue samples were randomly extracted and measured by Western blotting. ( c ) The results of quantitative analysis of NDUFA4L2 were significant. ( d ) The expression of NDUFA4L2 and HIF‐1α was measured by immunohistochemical staining. A high percentage of tumor cells and high staining intensity indicate high expression. Pneumocytes were the normal cells. ( e ) HIF‐1α and NDUFA4L2 protein levels in NSCLC cell lines were measured by Western blotting assays. ( f–i ) The results of quantitative analysis of NDUFA4L2 and HIF‐1α were significant. ( ) Normoxia (NX), and ( ) Hypoxia (HY). ( j ) Wound healing assays in NSCLC cell lines cultured in normoxia and hypoxia are shown. All data were analyzed as the mean ± standard deviation from at least three independent experiments. * P
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    Roche quantitative real time pcr total rna
    Dysregulation of long non‐coding <t>RNA</t> lnc NEN 885 could significantly affect epithelial‐mesenchymal transition ( EMT ) in gastroenteropancreatic neuroendocrine tumor cells. A,B, Classical EMT markers E‐cadherin, N‐cadherin, Snail, and vimentin levels under lnc NEN 885 overexpression or silencing conditions in BON ‐1 and LCC ‐18 cells were evaluated with western blot analysis. C,D, Expressions of E‐cadherin, N‐cadherin, Snail, and vimentin under lnc NEN 885 overexpression or silencing conditions in BON ‐1 and LCC ‐18 cells were detected with quantitative real‐time <t>PCR</t> . ** P
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    tiangen biotech co real time pcr total rna
    The interaction between EIF3B and piR-823 in LX-2 cells. ( A ) We collected the <t>RNA-protein</t> solution from RNA pull-down experiment and Coomassie brilliant blue staining showed that the piR-823 channel had 1 additional protein band as compared to the control channel (red box, indicated by black arrow). ( B ) Western blot analysis to test the EIF3B combined with piR-823. ( C ) Real-time <t>PCR</t> to test the piR-823 RNA combined with EIF3B. Bars=means ±SEM. n=3, * P
    Real Time Pcr Total Rna, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 93/100, based on 350 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore quantitative real time pcr rt pcr total rna
    Evaluation of the GLI1 expression in human melanoma cell lines and human primary melanoma tissues. A ) Relative GLI1 <t>RNA</t> expression in human melanoma cell lines and normal human melanocytes as measured by quantitative <t>RT-PCR.</t> B ) GLI1 RNA expression in human primary melanoma tissues harboring wild type (n = 7) or V600E activating mutation in BRAF (n = 15) evaluated by Affymetrix gene profiling. P
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    FUJIFILM real time pcr total rna
    Evaluation of the GLI1 expression in human melanoma cell lines and human primary melanoma tissues. A ) Relative GLI1 <t>RNA</t> expression in human melanoma cell lines and normal human melanocytes as measured by quantitative <t>RT-PCR.</t> B ) GLI1 RNA expression in human primary melanoma tissues harboring wild type (n = 7) or V600E activating mutation in BRAF (n = 15) evaluated by Affymetrix gene profiling. P
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    Millipore quantitative real time pcr total rna miniprep kit
    Evaluation of the GLI1 expression in human melanoma cell lines and human primary melanoma tissues. A ) Relative GLI1 <t>RNA</t> expression in human melanoma cell lines and normal human melanocytes as measured by quantitative <t>RT-PCR.</t> B ) GLI1 RNA expression in human primary melanoma tissues harboring wild type (n = 7) or V600E activating mutation in BRAF (n = 15) evaluated by Affymetrix gene profiling. P
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    iNtRON Biotechnology quantitative real time pcr total rna
    Expression of β-defensins in the lungs of WT and Nod1-dificient mice in fected with A. baumannii . <t>RNA</t> was obtained from the lungs of WT and Nod1-deficient mice (n=5–6 mice per group) infected with A. baumannii . The gene expressions of mBD-1, -2, -3, and -4 were examined by real-time <t>PCR</t> (A–D). Fold increase (arbitrary unit) was obtained by comparing each level in the lungs from infected to that in uninfected control lungs. * P
    Quantitative Real Time Pcr Total Rna, supplied by iNtRON Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher real time pcr qpcr total rna
    Overexpression of 14-3-3ε enhances HP-PRRSV infection. A Western blot analysis of 14-3-3β/ε overexpression cells. B Cell growth kinetics. Normal Marc-145 cells and Marc-145 wpxld , Marc-145 14-3-3β , and Marc-145 14-3-3ε cells were plated in 24-well plates. At 24, 48, 72, 96, 120, 144, and 168 h post-plating, the CCK-8 reagent was added to the cells. The cells were then incubated for 2 h at 37 °C. Cell counting was performed by measuring absorbance at 450 nm. Marc-145 14-3-3β , Marc-145 14-3-3ε , and Marc-145 wpxld cells were infected with TA-12 and CH-1R, respectively. The infected cells were harvested at 0, 6, 12, 24, 36, and 48 hpi for <t>RNA</t> extraction and at 12, 24, 36, and 48 hpi for protein extraction. Viral loads were evaluated by absolute <t>qPCR</t> targeting the N gene of TA-12 ( C ) and CH-1R ( D ). Cellular proteins were analyzed by Western blot analysis for detecting the viral N protein of TA-12 ( E ) and CH-1R ( F ). The GAPDH protein or GAPDH gene was used as the internal control. * P value
    Real Time Pcr Qpcr Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 304 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega quantitative real time pcr analysis total rna
    Overexpression of 14-3-3ε enhances HP-PRRSV infection. A Western blot analysis of 14-3-3β/ε overexpression cells. B Cell growth kinetics. Normal Marc-145 cells and Marc-145 wpxld , Marc-145 14-3-3β , and Marc-145 14-3-3ε cells were plated in 24-well plates. At 24, 48, 72, 96, 120, 144, and 168 h post-plating, the CCK-8 reagent was added to the cells. The cells were then incubated for 2 h at 37 °C. Cell counting was performed by measuring absorbance at 450 nm. Marc-145 14-3-3β , Marc-145 14-3-3ε , and Marc-145 wpxld cells were infected with TA-12 and CH-1R, respectively. The infected cells were harvested at 0, 6, 12, 24, 36, and 48 hpi for <t>RNA</t> extraction and at 12, 24, 36, and 48 hpi for protein extraction. Viral loads were evaluated by absolute <t>qPCR</t> targeting the N gene of TA-12 ( C ) and CH-1R ( D ). Cellular proteins were analyzed by Western blot analysis for detecting the viral N protein of TA-12 ( E ) and CH-1R ( F ). The GAPDH protein or GAPDH gene was used as the internal control. * P value
    Quantitative Real Time Pcr Analysis Total Rna, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa quantitative real time pcr analysis total rna
    Expression analysis of PtrANR1 and PtrLAR1 in P. trichocarpa tissues. (A) Semi-quantitative <t>RT-PCR</t> analysis of PtrANR1 and PtrLAR1 expression in various tissues of P. trichocarpa . (B) Quantitative real-time PCR analysis of PtrANR1 transcript levels in various tissues of P. trichocarpa. (C) Quantitative real-time PCR analysis of PtrLAR1 transcript levels in various tissues of P. trichocarpa . Poplar 18S expression was used as a control. Total <t>RNA</t> was isolated from roots (R), stems (S), petioles (P), mature leaves (ML), and young leaves (YL).
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    MACHEREY NAGEL real time pcr total rna extraction
    ETV5 and SOX9 do not regulate N-cadherin ( A ) Transcriptional regulators deregulated in the presence of AR-V7 compared to AR-WT with known binding sites in CDH2 gene are listed. ( B ) IPA analysis from deregulated genes in the presence of AR-V7 highlighted a network in which ETV5 was a direct regulator of CDH2. Green: downregulated expression; Red: upregulated expression. ( C ) A lentiviral inducible system was used to verify ETV5 expression in the presence of AR variants observed in our <t>RNA-seq</t> data. AR-WT and AR variants expression were induced with 20 ng/mL doxycycline. LNCaP expressing AR-WT and AR variants were cultured in the presence of 10nM DHT and ETV5 mRNA expression level was analyzed by real-time <t>PCR</t> 4 days after induction. ( D ) To analyze the impact of ETV5 on N-cadherin expression, AR-WT and AR-V7 were induced in LNCaP with 20 ng/mL doxycycline and cells were transfected with 50 nM of siRNA against ETV5 (left panel). After 48 h, total mRNA was extracted and CDH2 mRNA expression level was assessed by qRT-PCR (right panel). For all qRT-PCR analyses, the results were normalized to β-ACTIN. Relative expression is represented as the mean of ΔΔCt ± SEM of three independent experiments. NS: not significant, *** P
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    TaKaRa real time pcr assay total rna
    ETV5 and SOX9 do not regulate N-cadherin ( A ) Transcriptional regulators deregulated in the presence of AR-V7 compared to AR-WT with known binding sites in CDH2 gene are listed. ( B ) IPA analysis from deregulated genes in the presence of AR-V7 highlighted a network in which ETV5 was a direct regulator of CDH2. Green: downregulated expression; Red: upregulated expression. ( C ) A lentiviral inducible system was used to verify ETV5 expression in the presence of AR variants observed in our <t>RNA-seq</t> data. AR-WT and AR variants expression were induced with 20 ng/mL doxycycline. LNCaP expressing AR-WT and AR variants were cultured in the presence of 10nM DHT and ETV5 mRNA expression level was analyzed by real-time <t>PCR</t> 4 days after induction. ( D ) To analyze the impact of ETV5 on N-cadherin expression, AR-WT and AR-V7 were induced in LNCaP with 20 ng/mL doxycycline and cells were transfected with 50 nM of siRNA against ETV5 (left panel). After 48 h, total mRNA was extracted and CDH2 mRNA expression level was assessed by qRT-PCR (right panel). For all qRT-PCR analyses, the results were normalized to β-ACTIN. Relative expression is represented as the mean of ΔΔCt ± SEM of three independent experiments. NS: not significant, *** P
    Real Time Pcr Assay Total Rna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad quantitative real time pcr total rna
    DNMT3B modulates exon 4–5–6 skipping by physically interacting with hnRNP-LL and CD45 pre-mRNA. ( A ) Native <t>RNA</t> immunoprecipitation assay (RIP-qPCR) shows DNMT3B interaction with CD45 pre-mRNA in control and ICF1 samples. Binding to HOXC4 mRNA is reported as negative control; ( B ) DNMT3B enrichment at DNA (exons 4, 5 and 6) of CD45 gene by ChIP assay; ( C and D ) Expression level of hnRNP-LL in ICF1 samples and controls by qPCR and western blot, respectively; ( E ) CpG methylation level [log2 (methylated/unmethylated)] at hnRNP-LL promoter in ICF1 samples; ( F ) DNMT3B and hnRNP-LL physically interact in ICF1p2 sample as shown in co-immunoprecipitation experiments; ( G ) Expression level (qPCR) of CD45RABC and the constitutive exon9 using isoform specific oligonucleotides upon siDNMT3B and control siRNAs transfection; ( H ) Semi-quantitative <t>PCR</t> amplification of CD45 splicing isoforms in overexpressing-hnRNP-LL ICF1p2 sample upon siDNMT3B and control siRNAs transfection. * P -adj
    Quantitative Real Time Pcr Total Rna, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 161 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Genematrix real time pcr analysis total rna
    DNMT3B modulates exon 4–5–6 skipping by physically interacting with hnRNP-LL and CD45 pre-mRNA. ( A ) Native <t>RNA</t> immunoprecipitation assay (RIP-qPCR) shows DNMT3B interaction with CD45 pre-mRNA in control and ICF1 samples. Binding to HOXC4 mRNA is reported as negative control; ( B ) DNMT3B enrichment at DNA (exons 4, 5 and 6) of CD45 gene by ChIP assay; ( C and D ) Expression level of hnRNP-LL in ICF1 samples and controls by qPCR and western blot, respectively; ( E ) CpG methylation level [log2 (methylated/unmethylated)] at hnRNP-LL promoter in ICF1 samples; ( F ) DNMT3B and hnRNP-LL physically interact in ICF1p2 sample as shown in co-immunoprecipitation experiments; ( G ) Expression level (qPCR) of CD45RABC and the constitutive exon9 using isoform specific oligonucleotides upon siDNMT3B and control siRNAs transfection; ( H ) Semi-quantitative <t>PCR</t> amplification of CD45 splicing isoforms in overexpressing-hnRNP-LL ICF1p2 sample upon siDNMT3B and control siRNAs transfection. * P -adj
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    M6P/IGF2R expression increases during monocyte differentiation to macrophages . ( A ) Cell‐surface expression of M6P/IGF2R on isolated PBMC and MACS‐enriched monocytes from healthy donors was evaluated with mAb MEM‐238‐AF647 and flow cytometry. In parallel, MOPC‐21‐AF647 was used as an isotype control mAb, displayed by the cut‐off gates. The same analysis was performed with macrophages differentiated from MACS‐sorted monocytes during a 7‐day culture with recombinant human M‐CSF (50 ng/ml) followed by 2 days resting in macrophage serum free medium. ( B ) Primary human monocytes and monocyte‐derived macrophages from (A) were lysed and RNA was extracted. cDNA was synthesized from total RNA and gene expression was measured by real‐time PCR as described in the Material and Methods section with TaqMan primer sets for human M6P/IGF2R and YWHAZ as endogenous control. The M6P/IGF2R mean expression values relative to that of monocytes ± SD from 3 donors is shown

    Journal: Journal of Leukocyte Biology

    Article Title: The mannose 6‐phosphate/insulin‐like growth factor 2 receptor mediates plasminogen‐induced efferocytosis, et al. The mannose 6‐phosphate/insulin‐like growth factor 2 receptor mediates plasminogen‐induced efferocytosis

    doi: 10.1002/JLB.1AB0417-160RR

    Figure Lengend Snippet: M6P/IGF2R expression increases during monocyte differentiation to macrophages . ( A ) Cell‐surface expression of M6P/IGF2R on isolated PBMC and MACS‐enriched monocytes from healthy donors was evaluated with mAb MEM‐238‐AF647 and flow cytometry. In parallel, MOPC‐21‐AF647 was used as an isotype control mAb, displayed by the cut‐off gates. The same analysis was performed with macrophages differentiated from MACS‐sorted monocytes during a 7‐day culture with recombinant human M‐CSF (50 ng/ml) followed by 2 days resting in macrophage serum free medium. ( B ) Primary human monocytes and monocyte‐derived macrophages from (A) were lysed and RNA was extracted. cDNA was synthesized from total RNA and gene expression was measured by real‐time PCR as described in the Material and Methods section with TaqMan primer sets for human M6P/IGF2R and YWHAZ as endogenous control. The M6P/IGF2R mean expression values relative to that of monocytes ± SD from 3 donors is shown

    Article Snippet: 2.6 RNA isolation and real‐time PCR Total RNA was extracted from the cells with TRIzol reagent (Invitrogen) supplemented with β‐mercaptoethanol for RNAse inhibition. cDNA was synthesized from 500 ng total RNA using SuperScript III reverse transcriptase (Invitrogen).

    Techniques: Expressing, Isolation, Magnetic Cell Separation, Flow Cytometry, Cytometry, Recombinant, Derivative Assay, Synthesized, Real-time Polymerase Chain Reaction

    IgSF11 regulates in vitro osteoclast differentiation. a Osteoclast differentiation of IgSF11 +/+ and IgSF11 −/− cells. BMMs were cultured with M-CSF + RANKL for the indicated days (top). TRAP activity and frequency of TRAP + multinucleated cells (3 nuclei or more per cell) are shown (bottom). The scale bar represents 100 μm. b Gene expression during osteoclast differentiation. Total RNA was collected from IgSF11 +/+ and IgSF11 −/− cultured cells, and the levels of the indicated genes were measured by Q-PCR. c Bone resorption activity of IgSF11 +/+ and IgSF11 −/− osteoclasts. IgSF11 +/+ and IgSF11 −/− BMMs were cultured with M-CSF + RANKL for three days, harvested, and recultured on dentin slices. The resorption area and pit are shown per cell. The scale bar represents 100 μm. d Osteoclast differentiation rescued by retroviral transduction of IgSF11 in IgSF11 −/− BMMs. BMMs were retrovirally transduced with empty vector (EV) or Flag-tagged IgSF11 expression vector followed by culture with M-CSF + RANKL for three days. The frequency of TRAP + multinucleated cells (3 nuclei or more per cell) is shown. Expression of exogenous IgSF11 was confirmed by western blotting with an anti-Flag antibody. The scale bar represents 100 μm. Data are shown as the mean ± S.D. *** P

    Journal: Bone Research

    Article Title: IgSF11 regulates osteoclast differentiation through association with the scaffold protein PSD-95

    doi: 10.1038/s41413-019-0080-9

    Figure Lengend Snippet: IgSF11 regulates in vitro osteoclast differentiation. a Osteoclast differentiation of IgSF11 +/+ and IgSF11 −/− cells. BMMs were cultured with M-CSF + RANKL for the indicated days (top). TRAP activity and frequency of TRAP + multinucleated cells (3 nuclei or more per cell) are shown (bottom). The scale bar represents 100 μm. b Gene expression during osteoclast differentiation. Total RNA was collected from IgSF11 +/+ and IgSF11 −/− cultured cells, and the levels of the indicated genes were measured by Q-PCR. c Bone resorption activity of IgSF11 +/+ and IgSF11 −/− osteoclasts. IgSF11 +/+ and IgSF11 −/− BMMs were cultured with M-CSF + RANKL for three days, harvested, and recultured on dentin slices. The resorption area and pit are shown per cell. The scale bar represents 100 μm. d Osteoclast differentiation rescued by retroviral transduction of IgSF11 in IgSF11 −/− BMMs. BMMs were retrovirally transduced with empty vector (EV) or Flag-tagged IgSF11 expression vector followed by culture with M-CSF + RANKL for three days. The frequency of TRAP + multinucleated cells (3 nuclei or more per cell) is shown. Expression of exogenous IgSF11 was confirmed by western blotting with an anti-Flag antibody. The scale bar represents 100 μm. Data are shown as the mean ± S.D. *** P

    Article Snippet: Reverse transcription and real-time PCR (Q-PCR) Total RNA was extracted from cells using TRIzol reagent (Invitrogen), and 1μg–5 μg of total RNA was reverse transcribed using random hexamer primers and SuperScript III reverse transcriptase (Invitrogen). cDNA corresponding to 10 ng of total RNA was analyzed by Q-PCR using a QuantStudio3 (Applied Biosystems) and the following specific TaqMan® probes: IgSF11 (Mm00464360_m1), ACP5 (Mm00475698_m1), DC-STAMP (Mm01168058_m1), Ctsk (Mm00484036_m1), PSD-95 (Mm00492193_m1), and 18 S (Hs99999901_s1).

    Techniques: In Vitro, Cell Culture, Activity Assay, Expressing, Polymerase Chain Reaction, Transduction, Plasmid Preparation, Western Blot

    Identification of IgSF11 as an osteoclast differentiation-associated gene. a IgSF11 message expression during osteoclast differentiation. Total RNA was isolated from BMMs cultured with M-CSF + RANKL for the indicated days and used for Q-PCR. b IgSF11 protein expression during osteoclast differentiation. Total cell lysates were prepared from BMMs cultured with M-CSF + RANKL for the indicated days and used for western blotting with the indicated antibodies. c Effect of IgSF11 RNAi on osteoclast differentiation. BMMs retrovirally transduced with the indicated shRNAs were cultured with M-CSF + RANKL for three days. Relative expression of DC-STAMP and IgSF11 was determined by Q-PCR (left). Cells were stained for TRAP (middle). The frequency of TRAP + multinucleated cells is shown (right). The scale bar represents 100 μm. d Effect of antibodies on osteoclast differentiation. BMMs were cultured with M-CSF + RANKL for three days in the presence of the indicated antibodies (left). The frequency of TRAP + multinucleated cells (3 nuclei or more per cell) is shown (right). The scale bar represents 100 μm. Data are shown as the mean ± S.D. *** P

    Journal: Bone Research

    Article Title: IgSF11 regulates osteoclast differentiation through association with the scaffold protein PSD-95

    doi: 10.1038/s41413-019-0080-9

    Figure Lengend Snippet: Identification of IgSF11 as an osteoclast differentiation-associated gene. a IgSF11 message expression during osteoclast differentiation. Total RNA was isolated from BMMs cultured with M-CSF + RANKL for the indicated days and used for Q-PCR. b IgSF11 protein expression during osteoclast differentiation. Total cell lysates were prepared from BMMs cultured with M-CSF + RANKL for the indicated days and used for western blotting with the indicated antibodies. c Effect of IgSF11 RNAi on osteoclast differentiation. BMMs retrovirally transduced with the indicated shRNAs were cultured with M-CSF + RANKL for three days. Relative expression of DC-STAMP and IgSF11 was determined by Q-PCR (left). Cells were stained for TRAP (middle). The frequency of TRAP + multinucleated cells is shown (right). The scale bar represents 100 μm. d Effect of antibodies on osteoclast differentiation. BMMs were cultured with M-CSF + RANKL for three days in the presence of the indicated antibodies (left). The frequency of TRAP + multinucleated cells (3 nuclei or more per cell) is shown (right). The scale bar represents 100 μm. Data are shown as the mean ± S.D. *** P

    Article Snippet: Reverse transcription and real-time PCR (Q-PCR) Total RNA was extracted from cells using TRIzol reagent (Invitrogen), and 1μg–5 μg of total RNA was reverse transcribed using random hexamer primers and SuperScript III reverse transcriptase (Invitrogen). cDNA corresponding to 10 ng of total RNA was analyzed by Q-PCR using a QuantStudio3 (Applied Biosystems) and the following specific TaqMan® probes: IgSF11 (Mm00464360_m1), ACP5 (Mm00475698_m1), DC-STAMP (Mm01168058_m1), Ctsk (Mm00484036_m1), PSD-95 (Mm00492193_m1), and 18 S (Hs99999901_s1).

    Techniques: Expressing, Isolation, Cell Culture, Polymerase Chain Reaction, Western Blot, Transduction, Staining

    RPKM of putative ELO, FAR and WS genes. A, RPKM of 6 ELO genes; B, RPKM of 43 FAR genes; C, RPKM of 26 enzymes of acyltransferase family, green bars represent the WS genes; D, RPKM of 44 ABC transporters genes; E, RPKM of 113 putative WS genes through BLAST search using bio-cloud computing. Red bars of each histogram represent the genes with the highest RPKM and are selected for qRT-PCR analysis.

    Journal: PLoS ONE

    Article Title: Transcriptome Analysis of the Chinese White Wax Scale Ericerus pela with Focus on Genes Involved in Wax Biosynthesis

    doi: 10.1371/journal.pone.0035719

    Figure Lengend Snippet: RPKM of putative ELO, FAR and WS genes. A, RPKM of 6 ELO genes; B, RPKM of 43 FAR genes; C, RPKM of 26 enzymes of acyltransferase family, green bars represent the WS genes; D, RPKM of 44 ABC transporters genes; E, RPKM of 113 putative WS genes through BLAST search using bio-cloud computing. Red bars of each histogram represent the genes with the highest RPKM and are selected for qRT-PCR analysis.

    Article Snippet: Quantitative real-time PCR (qRT-PCR) analysis Total RNA of CWWS at different development stages was extracted separately, and 2 µg total RNA of each sample was reverse-transcribed in a 20 µl reaction system according to the protocol provided with the M-MLV first strand kit (Invitrogen, China).

    Techniques: Quantitative RT-PCR

    HS-induced transcription of Hsp70, Hsp83 and Hsp22 genes is decreased in Chd1 -deficient and Chd1 C D -rescued larvae. Larvae bearing the indicated transgenes in a Chd1 2 /Exel7014 genetic background and wild-type larvae were subjected to the indicated periods of HS before RNA was prepared and expression of the HS genes was analyzed by real-time PCR. All values were normalized against the non-HS (0) sample of the respective fly strain. Error bars indicate standard deviations of three independent experiments. ( A ) Hsp70 , ( B ) Hsp83 and ( C ) Hsp22 .

    Journal: Nucleic Acids Research

    Article Title: The chromodomains of CHD1 are critical for enzymatic activity but less important for chromatin localization

    doi: 10.1093/nar/gkq1298

    Figure Lengend Snippet: HS-induced transcription of Hsp70, Hsp83 and Hsp22 genes is decreased in Chd1 -deficient and Chd1 C D -rescued larvae. Larvae bearing the indicated transgenes in a Chd1 2 /Exel7014 genetic background and wild-type larvae were subjected to the indicated periods of HS before RNA was prepared and expression of the HS genes was analyzed by real-time PCR. All values were normalized against the non-HS (0) sample of the respective fly strain. Error bars indicate standard deviations of three independent experiments. ( A ) Hsp70 , ( B ) Hsp83 and ( C ) Hsp22 .

    Article Snippet: Real-time PCR analysis Total RNA was extracted by the TriBase (Sigma) method from larvae that had been subjected to various times (0, 5, 10, 15, 30 and 60 min) of heat treatment at 37°C and subsequently been frozen in liquid nitrogen.

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    HMMR-AS1 stabilizes HMMR mRNA. (A) Relative levels of HMMR-AS1 and HMMR mRNA in U87 and HA cells transfected with lentivirus containing HMMR-AS1 full-length sequence compare with control. (B) HMMR protein expression tested by Western blot in cells that HMMR-AS1 was downregulated or upregulated. (C) HMMR protein expression tested by immunofluorescence in control cells or cells with HMMR-AS1 or HMMR overexpression. Scale bar = 10 μm. (D) U87 cells differently transfected were treated with 50 μM α-amanitin for 6, 12, or 18 hours. Cells were then harvested for qRT-PCR to test the loss of HMMR mRNA. 18s RNA, a product of RNA polymerase I, was not affected by α-amanitin. Cells transfected with HMMR shRNA were used as positive control. β-Actin was used as a loading control for Western blot. The blot bands were quantified by ImageJ and represented by relative values compare with loading control (1.00). # P > .05, * P

    Journal: Neoplasia (New York, N.Y.)

    Article Title: Targeting Long Noncoding RNA HMMR-AS1 Suppresses and Radiosensitizes Glioblastoma

    doi: 10.1016/j.neo.2018.02.010

    Figure Lengend Snippet: HMMR-AS1 stabilizes HMMR mRNA. (A) Relative levels of HMMR-AS1 and HMMR mRNA in U87 and HA cells transfected with lentivirus containing HMMR-AS1 full-length sequence compare with control. (B) HMMR protein expression tested by Western blot in cells that HMMR-AS1 was downregulated or upregulated. (C) HMMR protein expression tested by immunofluorescence in control cells or cells with HMMR-AS1 or HMMR overexpression. Scale bar = 10 μm. (D) U87 cells differently transfected were treated with 50 μM α-amanitin for 6, 12, or 18 hours. Cells were then harvested for qRT-PCR to test the loss of HMMR mRNA. 18s RNA, a product of RNA polymerase I, was not affected by α-amanitin. Cells transfected with HMMR shRNA were used as positive control. β-Actin was used as a loading control for Western blot. The blot bands were quantified by ImageJ and represented by relative values compare with loading control (1.00). # P > .05, * P

    Article Snippet: Quantitative Real-Time PCR Assay Total RNA was extracted from samples by using Trizol (Invitrogen), and then the RNA was reverse transcribed to form cDNA by SuperScript III Reverse Transcriptase (Invitrogen) as we described previously .

    Techniques: Transfection, Sequencing, Expressing, Western Blot, Immunofluorescence, Over Expression, Quantitative RT-PCR, shRNA, Positive Control

    Quantitative RT-PCR analysis of the expression of β-LCY1 , β-LCY2 , and β-CHX genes in the flavedo and the pulp of Navel orange ( C. sinensis ) (black bars) and Star Ruby grapefruit ( C. paradisi ) (white bars) at the IG (immature green), MG (mature green), B (breaker), and FC (full-colour) stages. The levels of expression were normalized to the amount of RNA and the value of Navel flavedo at the FC stage was set to 100. The data are means ±SD of three experimental replicates.

    Journal: Journal of Experimental Botany

    Article Title: Molecular and functional characterization of a novel chromoplast-specific lycopene ?-cyclase from Citrus and its relation to lycopene accumulation

    doi: 10.1093/jxb/erp048

    Figure Lengend Snippet: Quantitative RT-PCR analysis of the expression of β-LCY1 , β-LCY2 , and β-CHX genes in the flavedo and the pulp of Navel orange ( C. sinensis ) (black bars) and Star Ruby grapefruit ( C. paradisi ) (white bars) at the IG (immature green), MG (mature green), B (breaker), and FC (full-colour) stages. The levels of expression were normalized to the amount of RNA and the value of Navel flavedo at the FC stage was set to 100. The data are means ±SD of three experimental replicates.

    Article Snippet: Quantitative real-time PCR analysis Total RNA was treated with DNase (Ambion, Huntingdon, UK) and accurately quantified by fluorometric assay with the RiboGreen dye (Molecular Probes) following the manufacturer's instructions, in order to normalize mRNA levels as described by .

    Techniques: Quantitative RT-PCR, Expressing

    NDUFA4L2 is involved in the survival and migration of non‐small cell lung cancer (NSCLC) in hypoxia. ( a ) NDUFA4L2 messenger RNA (mRNA) of human NSCLC tissue samples was measured by quantitative PCR. ( b ) Four pairs of tissue samples were randomly extracted and measured by Western blotting. ( c ) The results of quantitative analysis of NDUFA4L2 were significant. ( d ) The expression of NDUFA4L2 and HIF‐1α was measured by immunohistochemical staining. A high percentage of tumor cells and high staining intensity indicate high expression. Pneumocytes were the normal cells. ( e ) HIF‐1α and NDUFA4L2 protein levels in NSCLC cell lines were measured by Western blotting assays. ( f–i ) The results of quantitative analysis of NDUFA4L2 and HIF‐1α were significant. ( ) Normoxia (NX), and ( ) Hypoxia (HY). ( j ) Wound healing assays in NSCLC cell lines cultured in normoxia and hypoxia are shown. All data were analyzed as the mean ± standard deviation from at least three independent experiments. * P

    Journal: Thoracic Cancer

    Article Title: Mitochondrial NDUFA4L2 protein promotes the vitality of lung cancer cells by repressing oxidative stress

    doi: 10.1111/1759-7714.12984

    Figure Lengend Snippet: NDUFA4L2 is involved in the survival and migration of non‐small cell lung cancer (NSCLC) in hypoxia. ( a ) NDUFA4L2 messenger RNA (mRNA) of human NSCLC tissue samples was measured by quantitative PCR. ( b ) Four pairs of tissue samples were randomly extracted and measured by Western blotting. ( c ) The results of quantitative analysis of NDUFA4L2 were significant. ( d ) The expression of NDUFA4L2 and HIF‐1α was measured by immunohistochemical staining. A high percentage of tumor cells and high staining intensity indicate high expression. Pneumocytes were the normal cells. ( e ) HIF‐1α and NDUFA4L2 protein levels in NSCLC cell lines were measured by Western blotting assays. ( f–i ) The results of quantitative analysis of NDUFA4L2 and HIF‐1α were significant. ( ) Normoxia (NX), and ( ) Hypoxia (HY). ( j ) Wound healing assays in NSCLC cell lines cultured in normoxia and hypoxia are shown. All data were analyzed as the mean ± standard deviation from at least three independent experiments. * P

    Article Snippet: Quantitative real‐time PCR Total RNA was isolated from NSCLC tissue samples and cell lines with TRIzol (D9108, Takara, Dalian, China) according to manufacturer protocol.

    Techniques: Migration, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Immunohistochemistry, Staining, Cell Culture, Standard Deviation

    Dysregulation of long non‐coding RNA lnc NEN 885 could significantly affect epithelial‐mesenchymal transition ( EMT ) in gastroenteropancreatic neuroendocrine tumor cells. A,B, Classical EMT markers E‐cadherin, N‐cadherin, Snail, and vimentin levels under lnc NEN 885 overexpression or silencing conditions in BON ‐1 and LCC ‐18 cells were evaluated with western blot analysis. C,D, Expressions of E‐cadherin, N‐cadherin, Snail, and vimentin under lnc NEN 885 overexpression or silencing conditions in BON ‐1 and LCC ‐18 cells were detected with quantitative real‐time PCR . ** P

    Journal: Cancer Science

    Article Title: LncNEN885 inhibits epithelial‐mesenchymal transition by partially regulation of Wnt/β‐catenin signalling in gastroenteropancreatic neuroendocrine neoplasms, et al. LncNEN885 inhibits epithelial‐mesenchymal transition by partially regulation of Wnt/β‐catenin signalling in gastroenteropancreatic neuroendocrine neoplasms

    doi: 10.1111/cas.13747

    Figure Lengend Snippet: Dysregulation of long non‐coding RNA lnc NEN 885 could significantly affect epithelial‐mesenchymal transition ( EMT ) in gastroenteropancreatic neuroendocrine tumor cells. A,B, Classical EMT markers E‐cadherin, N‐cadherin, Snail, and vimentin levels under lnc NEN 885 overexpression or silencing conditions in BON ‐1 and LCC ‐18 cells were evaluated with western blot analysis. C,D, Expressions of E‐cadherin, N‐cadherin, Snail, and vimentin under lnc NEN 885 overexpression or silencing conditions in BON ‐1 and LCC ‐18 cells were detected with quantitative real‐time PCR . ** P

    Article Snippet: 2.5 RNA extraction and quantitative real‐time PCR Total RNA was extracted from cultured cells or tissues with TriPure Isolation Reagent (Roche, Penzberg, Germany) according to the manufacturer's instructions.

    Techniques: Over Expression, Western Blot, Real-time Polymerase Chain Reaction

    Long non‐coding RNA lnc NEN 885 could significantly inhibit Wnt/β‐catenin signaling. A, Markers of classical Wnt/β‐catenin signaling glycogen synthase kinase 3β ( GSK 3β), phosphorylated (p‐) GSK 3β, Axin, adenomatous polyposis coli ( APC ), and β‐catenin levels under lnc NEN 885 overexpression or silencing conditions in BON ‐1 cells were detected with western blotting. B, Expressions of GSK 3β, p‐ GSK 3β, Axin, APC , and β‐catenin under lnc NEN 885 overexpression or silencing conditions in LCC ‐18 cells were detected with western blotting. C,D, Expressions of GSK 3β, p‐ GSK 3β, Axin, APC , and β‐catenin under lnc NEN 885 overexpression or silencing conditions in BON ‐1 and LCC ‐18 cells were detected with quantitative real‐time PCR assay. ** P

    Journal: Cancer Science

    Article Title: LncNEN885 inhibits epithelial‐mesenchymal transition by partially regulation of Wnt/β‐catenin signalling in gastroenteropancreatic neuroendocrine neoplasms, et al. LncNEN885 inhibits epithelial‐mesenchymal transition by partially regulation of Wnt/β‐catenin signalling in gastroenteropancreatic neuroendocrine neoplasms

    doi: 10.1111/cas.13747

    Figure Lengend Snippet: Long non‐coding RNA lnc NEN 885 could significantly inhibit Wnt/β‐catenin signaling. A, Markers of classical Wnt/β‐catenin signaling glycogen synthase kinase 3β ( GSK 3β), phosphorylated (p‐) GSK 3β, Axin, adenomatous polyposis coli ( APC ), and β‐catenin levels under lnc NEN 885 overexpression or silencing conditions in BON ‐1 cells were detected with western blotting. B, Expressions of GSK 3β, p‐ GSK 3β, Axin, APC , and β‐catenin under lnc NEN 885 overexpression or silencing conditions in LCC ‐18 cells were detected with western blotting. C,D, Expressions of GSK 3β, p‐ GSK 3β, Axin, APC , and β‐catenin under lnc NEN 885 overexpression or silencing conditions in BON ‐1 and LCC ‐18 cells were detected with quantitative real‐time PCR assay. ** P

    Article Snippet: 2.5 RNA extraction and quantitative real‐time PCR Total RNA was extracted from cultured cells or tissues with TriPure Isolation Reagent (Roche, Penzberg, Germany) according to the manufacturer's instructions.

    Techniques: Over Expression, Western Blot, Real-time Polymerase Chain Reaction

    Long non‐coding RNA lnc NEN 885‐related epithelial‐mesenchymal transition ( EMT ) might partially rely on Wnt/β‐catenin signaling. A,B, Classical EMT markers E‐cadherin, N‐cadherin, Snail, and vimentin levels under siRNA (si)‐β‐catenin or cotransfection with si‐β‐catenin and lentivirus (Lv)‐Lnc NEN 885 or si‐Lnc NEN 885 conditions in BON ‐1 and LCC ‐18 cells were evaluated with western blotting. C,D, Expressions of classical EMT markers E‐cadherin, N‐cadherin, Snail, and vimentin levels under si‐β‐catenin or cotransfection with si‐β‐catenin and Lv‐Lnc NEN 885 or si‐Lnc NEN 885 conditions in BON ‐1 and LCC ‐18 cells were detected with quantitative real‐time PCR assay. ** P

    Journal: Cancer Science

    Article Title: LncNEN885 inhibits epithelial‐mesenchymal transition by partially regulation of Wnt/β‐catenin signalling in gastroenteropancreatic neuroendocrine neoplasms, et al. LncNEN885 inhibits epithelial‐mesenchymal transition by partially regulation of Wnt/β‐catenin signalling in gastroenteropancreatic neuroendocrine neoplasms

    doi: 10.1111/cas.13747

    Figure Lengend Snippet: Long non‐coding RNA lnc NEN 885‐related epithelial‐mesenchymal transition ( EMT ) might partially rely on Wnt/β‐catenin signaling. A,B, Classical EMT markers E‐cadherin, N‐cadherin, Snail, and vimentin levels under siRNA (si)‐β‐catenin or cotransfection with si‐β‐catenin and lentivirus (Lv)‐Lnc NEN 885 or si‐Lnc NEN 885 conditions in BON ‐1 and LCC ‐18 cells were evaluated with western blotting. C,D, Expressions of classical EMT markers E‐cadherin, N‐cadherin, Snail, and vimentin levels under si‐β‐catenin or cotransfection with si‐β‐catenin and Lv‐Lnc NEN 885 or si‐Lnc NEN 885 conditions in BON ‐1 and LCC ‐18 cells were detected with quantitative real‐time PCR assay. ** P

    Article Snippet: 2.5 RNA extraction and quantitative real‐time PCR Total RNA was extracted from cultured cells or tissues with TriPure Isolation Reagent (Roche, Penzberg, Germany) according to the manufacturer's instructions.

    Techniques: Cotransfection, Western Blot, Real-time Polymerase Chain Reaction

    Long non‐coding RNA (lnc RNA ) expression profiles and efficiency of lentivirus (Lv)‐lnc NEN 885 or siRNA (si)‐lnc NEN 885. A, Gene chip analysis of lnc RNA s transcripts in gastroenteropancreatic neuroendocrine neoplasms ( GEP ‐ NEN s) and adjacent normal tissues. B, Quantitative real‐time PCR analysis of lnc NEN 885 expression in GEP ‐ NEN s and adjacent normal tissues (N). T, tumor samples. C, Efficiency of Lv‐lnc NEN 885 and Lv‐control in BON ‐1 and LCC ‐18 cells (Lv‐, lentivirus, overexpression). D, Efficiency of three pairs of si‐lnc NEN 885 compared si‐control in BON ‐1 and LCC ‐18 cells (si‐, si RNA , silencing). Lnc NEN 885 expression was significantly downregulated in the si RNA group compared with the si‐control group, especially the si‐Lnc NEN 885‐1 group. Thus, si‐Lnc NEN 885‐1 was chosen for the following experiments, abbreviated as si‐Lnc NEN 885. ** P

    Journal: Cancer Science

    Article Title: LncNEN885 inhibits epithelial‐mesenchymal transition by partially regulation of Wnt/β‐catenin signalling in gastroenteropancreatic neuroendocrine neoplasms, et al. LncNEN885 inhibits epithelial‐mesenchymal transition by partially regulation of Wnt/β‐catenin signalling in gastroenteropancreatic neuroendocrine neoplasms

    doi: 10.1111/cas.13747

    Figure Lengend Snippet: Long non‐coding RNA (lnc RNA ) expression profiles and efficiency of lentivirus (Lv)‐lnc NEN 885 or siRNA (si)‐lnc NEN 885. A, Gene chip analysis of lnc RNA s transcripts in gastroenteropancreatic neuroendocrine neoplasms ( GEP ‐ NEN s) and adjacent normal tissues. B, Quantitative real‐time PCR analysis of lnc NEN 885 expression in GEP ‐ NEN s and adjacent normal tissues (N). T, tumor samples. C, Efficiency of Lv‐lnc NEN 885 and Lv‐control in BON ‐1 and LCC ‐18 cells (Lv‐, lentivirus, overexpression). D, Efficiency of three pairs of si‐lnc NEN 885 compared si‐control in BON ‐1 and LCC ‐18 cells (si‐, si RNA , silencing). Lnc NEN 885 expression was significantly downregulated in the si RNA group compared with the si‐control group, especially the si‐Lnc NEN 885‐1 group. Thus, si‐Lnc NEN 885‐1 was chosen for the following experiments, abbreviated as si‐Lnc NEN 885. ** P

    Article Snippet: 2.5 RNA extraction and quantitative real‐time PCR Total RNA was extracted from cultured cells or tissues with TriPure Isolation Reagent (Roche, Penzberg, Germany) according to the manufacturer's instructions.

    Techniques: RNA Expression, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Expressing, Over Expression

    The interaction between EIF3B and piR-823 in LX-2 cells. ( A ) We collected the RNA-protein solution from RNA pull-down experiment and Coomassie brilliant blue staining showed that the piR-823 channel had 1 additional protein band as compared to the control channel (red box, indicated by black arrow). ( B ) Western blot analysis to test the EIF3B combined with piR-823. ( C ) Real-time PCR to test the piR-823 RNA combined with EIF3B. Bars=means ±SEM. n=3, * P

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: The Combination of piR-823 and Eukaryotic Initiation Factor 3 B (EIF3B) Activates Hepatic Stellate Cells via Upregulating TGF-β1 in Liver Fibrogenesis

    doi: 10.12659/MSM.914222

    Figure Lengend Snippet: The interaction between EIF3B and piR-823 in LX-2 cells. ( A ) We collected the RNA-protein solution from RNA pull-down experiment and Coomassie brilliant blue staining showed that the piR-823 channel had 1 additional protein band as compared to the control channel (red box, indicated by black arrow). ( B ) Western blot analysis to test the EIF3B combined with piR-823. ( C ) Real-time PCR to test the piR-823 RNA combined with EIF3B. Bars=means ±SEM. n=3, * P

    Article Snippet: Isolation of RNA and real-time PCR Total RNA was extracted using a Pure Cell/Bacteria kit (Tiangen, Beijing, China).

    Techniques: Staining, Western Blot, Real-time Polymerase Chain Reaction

    Evaluation of the GLI1 expression in human melanoma cell lines and human primary melanoma tissues. A ) Relative GLI1 RNA expression in human melanoma cell lines and normal human melanocytes as measured by quantitative RT-PCR. B ) GLI1 RNA expression in human primary melanoma tissues harboring wild type (n = 7) or V600E activating mutation in BRAF (n = 15) evaluated by Affymetrix gene profiling. P

    Journal: PLoS ONE

    Article Title: NVP-LDE225, a Potent and Selective SMOOTHENED Antagonist Reduces Melanoma Growth In Vitro and In Vivo

    doi: 10.1371/journal.pone.0069064

    Figure Lengend Snippet: Evaluation of the GLI1 expression in human melanoma cell lines and human primary melanoma tissues. A ) Relative GLI1 RNA expression in human melanoma cell lines and normal human melanocytes as measured by quantitative RT-PCR. B ) GLI1 RNA expression in human primary melanoma tissues harboring wild type (n = 7) or V600E activating mutation in BRAF (n = 15) evaluated by Affymetrix gene profiling. P

    Article Snippet: RNA isolation, gene expression profiling and quantitative real-time PCR (RT-PCR) analysis Total RNA was isolated from monolayer cell cultures and cryopreserved tissues using TriReagent (Sigma-Aldrich) according to the manufacturer's instructions.

    Techniques: Expressing, RNA Expression, Quantitative RT-PCR, Mutagenesis

    Expression of β-defensins in the lungs of WT and Nod1-dificient mice in fected with A. baumannii . RNA was obtained from the lungs of WT and Nod1-deficient mice (n=5–6 mice per group) infected with A. baumannii . The gene expressions of mBD-1, -2, -3, and -4 were examined by real-time PCR (A–D). Fold increase (arbitrary unit) was obtained by comparing each level in the lungs from infected to that in uninfected control lungs. * P

    Journal: Laboratory Animal Research

    Article Title: Nucleotide-binding oligomerization domain 1 is dispensable for host immune responses against pulmonary infection of Acinetobacter baumannii in mice

    doi: 10.5625/lar.2018.34.4.295

    Figure Lengend Snippet: Expression of β-defensins in the lungs of WT and Nod1-dificient mice in fected with A. baumannii . RNA was obtained from the lungs of WT and Nod1-deficient mice (n=5–6 mice per group) infected with A. baumannii . The gene expressions of mBD-1, -2, -3, and -4 were examined by real-time PCR (A–D). Fold increase (arbitrary unit) was obtained by comparing each level in the lungs from infected to that in uninfected control lungs. * P

    Article Snippet: Quantitative real-time PCR Total RNA was extracted from lung tissue using easy-BLUE (Intron Biotechnology, Korea) according to the manufacturer's instructions.

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

    Overexpression of 14-3-3ε enhances HP-PRRSV infection. A Western blot analysis of 14-3-3β/ε overexpression cells. B Cell growth kinetics. Normal Marc-145 cells and Marc-145 wpxld , Marc-145 14-3-3β , and Marc-145 14-3-3ε cells were plated in 24-well plates. At 24, 48, 72, 96, 120, 144, and 168 h post-plating, the CCK-8 reagent was added to the cells. The cells were then incubated for 2 h at 37 °C. Cell counting was performed by measuring absorbance at 450 nm. Marc-145 14-3-3β , Marc-145 14-3-3ε , and Marc-145 wpxld cells were infected with TA-12 and CH-1R, respectively. The infected cells were harvested at 0, 6, 12, 24, 36, and 48 hpi for RNA extraction and at 12, 24, 36, and 48 hpi for protein extraction. Viral loads were evaluated by absolute qPCR targeting the N gene of TA-12 ( C ) and CH-1R ( D ). Cellular proteins were analyzed by Western blot analysis for detecting the viral N protein of TA-12 ( E ) and CH-1R ( F ). The GAPDH protein or GAPDH gene was used as the internal control. * P value

    Journal: Veterinary Research

    Article Title: 14-3-3ε acts as a proviral factor in highly pathogenic porcine reproductive and respiratory syndrome virus infection

    doi: 10.1186/s13567-019-0636-0

    Figure Lengend Snippet: Overexpression of 14-3-3ε enhances HP-PRRSV infection. A Western blot analysis of 14-3-3β/ε overexpression cells. B Cell growth kinetics. Normal Marc-145 cells and Marc-145 wpxld , Marc-145 14-3-3β , and Marc-145 14-3-3ε cells were plated in 24-well plates. At 24, 48, 72, 96, 120, 144, and 168 h post-plating, the CCK-8 reagent was added to the cells. The cells were then incubated for 2 h at 37 °C. Cell counting was performed by measuring absorbance at 450 nm. Marc-145 14-3-3β , Marc-145 14-3-3ε , and Marc-145 wpxld cells were infected with TA-12 and CH-1R, respectively. The infected cells were harvested at 0, 6, 12, 24, 36, and 48 hpi for RNA extraction and at 12, 24, 36, and 48 hpi for protein extraction. Viral loads were evaluated by absolute qPCR targeting the N gene of TA-12 ( C ) and CH-1R ( D ). Cellular proteins were analyzed by Western blot analysis for detecting the viral N protein of TA-12 ( E ) and CH-1R ( F ). The GAPDH protein or GAPDH gene was used as the internal control. * P value

    Article Snippet: Real-time PCR (qPCR) Total RNA was isolated from Marc-145 cells or PAMs using the GeneJET RNA Purification Kit (Thermo Scientific, Massachusetts, USA) and then reverse transcribed using the ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan) in accordance with the manufacturer’s instructions.

    Techniques: Over Expression, Infection, Western Blot, CCK-8 Assay, Incubation, Cell Counting, RNA Extraction, Protein Extraction, Real-time Polymerase Chain Reaction

    Difopein decreases HP-PRRSV infection in Marc-145 cells. A The cytotoxicity of difopein was determined by the CCK-8 assay. Monolayers of Marc-145 cells in 96-well plates were treated with difopein at different concentrations for 48 h, after which the CCK-8 reagent was added to each well. After incubation for 2 h, cell viability was evaluated by measuring absorbance at 450 nm. Marc-145 cells were inoculated with TA-12 and CH-1R and incubated for 1 h, after which the medium was replaced with maintenance medium containing 0.02 or 0.08 μg/mL difopein. The cells were collected 24 h later, and total RNA and cellular proteins were extracted. Viral loads were evaluated by absolute qPCR targeting the nucleocapsid ( N ) gene, and viral proteins were detected by Western blot analysis of TA-12- ( B ) and CH-1R- ( C ) infected cells. D CCID 50 analysis for titration of HP-PRRSV after difopein treatment. Marc-145 cells were seeded in 96-well plates. Virus supernatants were tenfold serially diluted (range 10 2 –10 10 ) and added to each well (at 100 μL per well) in eight repetitions. At 6 days post-infection, the numbers of cells in wells exhibiting cytopathic effects were counted, and CCID 50 was calculated by the Reed–Muench method. E Marc-145 cells were infected with TA-12 and treated with 0.08 or 0.1 μg/mL difopein at 24 hpi. After incubation for another 12 h, the cells were collected for RNA extraction and qPCR analysis. * P value

    Journal: Veterinary Research

    Article Title: 14-3-3ε acts as a proviral factor in highly pathogenic porcine reproductive and respiratory syndrome virus infection

    doi: 10.1186/s13567-019-0636-0

    Figure Lengend Snippet: Difopein decreases HP-PRRSV infection in Marc-145 cells. A The cytotoxicity of difopein was determined by the CCK-8 assay. Monolayers of Marc-145 cells in 96-well plates were treated with difopein at different concentrations for 48 h, after which the CCK-8 reagent was added to each well. After incubation for 2 h, cell viability was evaluated by measuring absorbance at 450 nm. Marc-145 cells were inoculated with TA-12 and CH-1R and incubated for 1 h, after which the medium was replaced with maintenance medium containing 0.02 or 0.08 μg/mL difopein. The cells were collected 24 h later, and total RNA and cellular proteins were extracted. Viral loads were evaluated by absolute qPCR targeting the nucleocapsid ( N ) gene, and viral proteins were detected by Western blot analysis of TA-12- ( B ) and CH-1R- ( C ) infected cells. D CCID 50 analysis for titration of HP-PRRSV after difopein treatment. Marc-145 cells were seeded in 96-well plates. Virus supernatants were tenfold serially diluted (range 10 2 –10 10 ) and added to each well (at 100 μL per well) in eight repetitions. At 6 days post-infection, the numbers of cells in wells exhibiting cytopathic effects were counted, and CCID 50 was calculated by the Reed–Muench method. E Marc-145 cells were infected with TA-12 and treated with 0.08 or 0.1 μg/mL difopein at 24 hpi. After incubation for another 12 h, the cells were collected for RNA extraction and qPCR analysis. * P value

    Article Snippet: Real-time PCR (qPCR) Total RNA was isolated from Marc-145 cells or PAMs using the GeneJET RNA Purification Kit (Thermo Scientific, Massachusetts, USA) and then reverse transcribed using the ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan) in accordance with the manufacturer’s instructions.

    Techniques: Infection, CCK-8 Assay, Incubation, Real-time Polymerase Chain Reaction, Western Blot, Titration, Endpoint Dilution Assay, RNA Extraction

    14 - 3 - 3ε knockdown and difopein treatment decrease HP-PRRSV infection in PAMs. A Results of qPCR for detection of 14 - 3 - 3ε after knockdown. PAMs were transfected with siRNA. The cells were collected at 24 h post-transfection, and total RNA was prepared for detecting the mRNA levels of 14 - 3 - 3ε . B Results of qPCR for analysis of TA-12 replication after 14 - 3 - 3ε knockdown. PAMs were transfected with siRNA and inoculated with TA-12 at 24 h post-transfection. The cells were collected, and total RNA was extracted. Viral loads were evaluated by absolute qPCR targeting the N gene of TA-12. C The cytotoxicity of difopein in PAMs was determined by the CCK-8 assay. PAMs were inoculated with TA-12 or CH-1R and incubated for 1 h, after which the medium was replaced with maintenance medium containing 0.02 or 0.08 μg/mL difopein. The cells were collected 24 h later, and total RNA was extracted. Viral loads were evaluated by absolute qPCR targeting the N gene, and viral proteins were detected by Western blot analysis of TA-12- ( D ) CH-1R- ( E ) infected cells. F Therapeutic role of difopein in TA-12 infection. PAMs were infected with TA-12 and treated with 0.02 or 0.08 μg/mL difopein at 24 hpi. The cells were incubated for another 12 h and then harvested for RNA extraction and qPCR analysis.

    Journal: Veterinary Research

    Article Title: 14-3-3ε acts as a proviral factor in highly pathogenic porcine reproductive and respiratory syndrome virus infection

    doi: 10.1186/s13567-019-0636-0

    Figure Lengend Snippet: 14 - 3 - 3ε knockdown and difopein treatment decrease HP-PRRSV infection in PAMs. A Results of qPCR for detection of 14 - 3 - 3ε after knockdown. PAMs were transfected with siRNA. The cells were collected at 24 h post-transfection, and total RNA was prepared for detecting the mRNA levels of 14 - 3 - 3ε . B Results of qPCR for analysis of TA-12 replication after 14 - 3 - 3ε knockdown. PAMs were transfected with siRNA and inoculated with TA-12 at 24 h post-transfection. The cells were collected, and total RNA was extracted. Viral loads were evaluated by absolute qPCR targeting the N gene of TA-12. C The cytotoxicity of difopein in PAMs was determined by the CCK-8 assay. PAMs were inoculated with TA-12 or CH-1R and incubated for 1 h, after which the medium was replaced with maintenance medium containing 0.02 or 0.08 μg/mL difopein. The cells were collected 24 h later, and total RNA was extracted. Viral loads were evaluated by absolute qPCR targeting the N gene, and viral proteins were detected by Western blot analysis of TA-12- ( D ) CH-1R- ( E ) infected cells. F Therapeutic role of difopein in TA-12 infection. PAMs were infected with TA-12 and treated with 0.02 or 0.08 μg/mL difopein at 24 hpi. The cells were incubated for another 12 h and then harvested for RNA extraction and qPCR analysis.

    Article Snippet: Real-time PCR (qPCR) Total RNA was isolated from Marc-145 cells or PAMs using the GeneJET RNA Purification Kit (Thermo Scientific, Massachusetts, USA) and then reverse transcribed using the ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan) in accordance with the manufacturer’s instructions.

    Techniques: Infection, Real-time Polymerase Chain Reaction, Transfection, CCK-8 Assay, Incubation, Western Blot, RNA Extraction

    Knockdown of 14 - 3 - 3ε decreases HP-PRRSV infection. A Results of qPCR for detection of 14 - 3 - 3β/ε after siRNA transfection. Marc-145 cells were transfected with siRNA. At 24 h post-transfection, the cells were collected, and total RNA was prepared for detecting the mRNA levels of 14 - 3 - 3β/ε . B Western blot analysis of 14-3-3β/ε expression after siRNA transfection. Marc-145 cells were transfected with siRNA. At 24 h post-transfection, the cells were collected, and cellular proteins were extracted for detecting 14-3-3β/ε proteins. C Marc-145 cells were transfected with siRNA and inoculated with TA-12 or mock infected with cell-culture medium at 24 h post-transfection. The mock-infected cells were collected at different time points after infection, and their viability was measured by the CCK-8 assay. D The TA-12-infected cells were collected at the same time points as the mock-infected cells and processed for total RNA extraction. Viral loads were evaluated by absolute qPCR targeting the nucleocapsid ( N ) gene of HP-PRRSV . E Viral proteins were evaluated by Western blot using a monoclonal antibody (6D10) targeting the PRRSV N protein. N.C: negative control. GAPDH: glyceraldehyde-3-phosphate dehydrogenase. The GAPDH gene and protein were used as internal controls for qPCR and Western blot. The density of the protein bands—measured with a fusion analysis software by using the VILBER lourmat imaging system (Fusion FX7, France)—was determined after subtracting the density of the GAPDH bands. * P value

    Journal: Veterinary Research

    Article Title: 14-3-3ε acts as a proviral factor in highly pathogenic porcine reproductive and respiratory syndrome virus infection

    doi: 10.1186/s13567-019-0636-0

    Figure Lengend Snippet: Knockdown of 14 - 3 - 3ε decreases HP-PRRSV infection. A Results of qPCR for detection of 14 - 3 - 3β/ε after siRNA transfection. Marc-145 cells were transfected with siRNA. At 24 h post-transfection, the cells were collected, and total RNA was prepared for detecting the mRNA levels of 14 - 3 - 3β/ε . B Western blot analysis of 14-3-3β/ε expression after siRNA transfection. Marc-145 cells were transfected with siRNA. At 24 h post-transfection, the cells were collected, and cellular proteins were extracted for detecting 14-3-3β/ε proteins. C Marc-145 cells were transfected with siRNA and inoculated with TA-12 or mock infected with cell-culture medium at 24 h post-transfection. The mock-infected cells were collected at different time points after infection, and their viability was measured by the CCK-8 assay. D The TA-12-infected cells were collected at the same time points as the mock-infected cells and processed for total RNA extraction. Viral loads were evaluated by absolute qPCR targeting the nucleocapsid ( N ) gene of HP-PRRSV . E Viral proteins were evaluated by Western blot using a monoclonal antibody (6D10) targeting the PRRSV N protein. N.C: negative control. GAPDH: glyceraldehyde-3-phosphate dehydrogenase. The GAPDH gene and protein were used as internal controls for qPCR and Western blot. The density of the protein bands—measured with a fusion analysis software by using the VILBER lourmat imaging system (Fusion FX7, France)—was determined after subtracting the density of the GAPDH bands. * P value

    Article Snippet: Real-time PCR (qPCR) Total RNA was isolated from Marc-145 cells or PAMs using the GeneJET RNA Purification Kit (Thermo Scientific, Massachusetts, USA) and then reverse transcribed using the ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan) in accordance with the manufacturer’s instructions.

    Techniques: Infection, Real-time Polymerase Chain Reaction, Transfection, Western Blot, Expressing, Cell Culture, CCK-8 Assay, RNA Extraction, Negative Control, Software, Imaging

    Expression analysis of PtrANR1 and PtrLAR1 in P. trichocarpa tissues. (A) Semi-quantitative RT-PCR analysis of PtrANR1 and PtrLAR1 expression in various tissues of P. trichocarpa . (B) Quantitative real-time PCR analysis of PtrANR1 transcript levels in various tissues of P. trichocarpa. (C) Quantitative real-time PCR analysis of PtrLAR1 transcript levels in various tissues of P. trichocarpa . Poplar 18S expression was used as a control. Total RNA was isolated from roots (R), stems (S), petioles (P), mature leaves (ML), and young leaves (YL).

    Journal: PLoS ONE

    Article Title: Isolation and Characterization of cDNAs Encoding Leucoanthocyanidin Reductase and Anthocyanidin Reductase from Populus trichocarpa

    doi: 10.1371/journal.pone.0064664

    Figure Lengend Snippet: Expression analysis of PtrANR1 and PtrLAR1 in P. trichocarpa tissues. (A) Semi-quantitative RT-PCR analysis of PtrANR1 and PtrLAR1 expression in various tissues of P. trichocarpa . (B) Quantitative real-time PCR analysis of PtrANR1 transcript levels in various tissues of P. trichocarpa. (C) Quantitative real-time PCR analysis of PtrLAR1 transcript levels in various tissues of P. trichocarpa . Poplar 18S expression was used as a control. Total RNA was isolated from roots (R), stems (S), petioles (P), mature leaves (ML), and young leaves (YL).

    Article Snippet: Semi-quantitative RT-PCR and Quantitative Real-time PCR Analysis Total RNA was extracted from leaves, roots, stems, and petioles of poplar plants and treated with DNase I (TaKaRa) according to the manufacturer’s instructions.

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

    ETV5 and SOX9 do not regulate N-cadherin ( A ) Transcriptional regulators deregulated in the presence of AR-V7 compared to AR-WT with known binding sites in CDH2 gene are listed. ( B ) IPA analysis from deregulated genes in the presence of AR-V7 highlighted a network in which ETV5 was a direct regulator of CDH2. Green: downregulated expression; Red: upregulated expression. ( C ) A lentiviral inducible system was used to verify ETV5 expression in the presence of AR variants observed in our RNA-seq data. AR-WT and AR variants expression were induced with 20 ng/mL doxycycline. LNCaP expressing AR-WT and AR variants were cultured in the presence of 10nM DHT and ETV5 mRNA expression level was analyzed by real-time PCR 4 days after induction. ( D ) To analyze the impact of ETV5 on N-cadherin expression, AR-WT and AR-V7 were induced in LNCaP with 20 ng/mL doxycycline and cells were transfected with 50 nM of siRNA against ETV5 (left panel). After 48 h, total mRNA was extracted and CDH2 mRNA expression level was assessed by qRT-PCR (right panel). For all qRT-PCR analyses, the results were normalized to β-ACTIN. Relative expression is represented as the mean of ΔΔCt ± SEM of three independent experiments. NS: not significant, *** P

    Journal: Oncotarget

    Article Title: Dual effects of constitutively active androgen receptor and full-length androgen receptor for N-cadherin regulation in prostate cancer

    doi: 10.18632/oncotarget.18270

    Figure Lengend Snippet: ETV5 and SOX9 do not regulate N-cadherin ( A ) Transcriptional regulators deregulated in the presence of AR-V7 compared to AR-WT with known binding sites in CDH2 gene are listed. ( B ) IPA analysis from deregulated genes in the presence of AR-V7 highlighted a network in which ETV5 was a direct regulator of CDH2. Green: downregulated expression; Red: upregulated expression. ( C ) A lentiviral inducible system was used to verify ETV5 expression in the presence of AR variants observed in our RNA-seq data. AR-WT and AR variants expression were induced with 20 ng/mL doxycycline. LNCaP expressing AR-WT and AR variants were cultured in the presence of 10nM DHT and ETV5 mRNA expression level was analyzed by real-time PCR 4 days after induction. ( D ) To analyze the impact of ETV5 on N-cadherin expression, AR-WT and AR-V7 were induced in LNCaP with 20 ng/mL doxycycline and cells were transfected with 50 nM of siRNA against ETV5 (left panel). After 48 h, total mRNA was extracted and CDH2 mRNA expression level was assessed by qRT-PCR (right panel). For all qRT-PCR analyses, the results were normalized to β-ACTIN. Relative expression is represented as the mean of ΔΔCt ± SEM of three independent experiments. NS: not significant, *** P

    Article Snippet: Quantitative real-time PCR Total RNA was isolated using NucleoSpin® RNA II assay (Macherey-Nagel) according to the manufacturer's procedure and 400 ng of total RNA were reverse transcribed using iScript kit (Bio-Rad).

    Techniques: Binding Assay, Indirect Immunoperoxidase Assay, Expressing, RNA Sequencing Assay, Cell Culture, Real-time Polymerase Chain Reaction, Transfection, Quantitative RT-PCR

    DNMT3B modulates exon 4–5–6 skipping by physically interacting with hnRNP-LL and CD45 pre-mRNA. ( A ) Native RNA immunoprecipitation assay (RIP-qPCR) shows DNMT3B interaction with CD45 pre-mRNA in control and ICF1 samples. Binding to HOXC4 mRNA is reported as negative control; ( B ) DNMT3B enrichment at DNA (exons 4, 5 and 6) of CD45 gene by ChIP assay; ( C and D ) Expression level of hnRNP-LL in ICF1 samples and controls by qPCR and western blot, respectively; ( E ) CpG methylation level [log2 (methylated/unmethylated)] at hnRNP-LL promoter in ICF1 samples; ( F ) DNMT3B and hnRNP-LL physically interact in ICF1p2 sample as shown in co-immunoprecipitation experiments; ( G ) Expression level (qPCR) of CD45RABC and the constitutive exon9 using isoform specific oligonucleotides upon siDNMT3B and control siRNAs transfection; ( H ) Semi-quantitative PCR amplification of CD45 splicing isoforms in overexpressing-hnRNP-LL ICF1p2 sample upon siDNMT3B and control siRNAs transfection. * P -adj

    Journal: Nucleic Acids Research

    Article Title: ICF-specific DNMT3B dysfunction interferes with intragenic regulation of mRNA transcription and alternative splicing

    doi: 10.1093/nar/gkx163

    Figure Lengend Snippet: DNMT3B modulates exon 4–5–6 skipping by physically interacting with hnRNP-LL and CD45 pre-mRNA. ( A ) Native RNA immunoprecipitation assay (RIP-qPCR) shows DNMT3B interaction with CD45 pre-mRNA in control and ICF1 samples. Binding to HOXC4 mRNA is reported as negative control; ( B ) DNMT3B enrichment at DNA (exons 4, 5 and 6) of CD45 gene by ChIP assay; ( C and D ) Expression level of hnRNP-LL in ICF1 samples and controls by qPCR and western blot, respectively; ( E ) CpG methylation level [log2 (methylated/unmethylated)] at hnRNP-LL promoter in ICF1 samples; ( F ) DNMT3B and hnRNP-LL physically interact in ICF1p2 sample as shown in co-immunoprecipitation experiments; ( G ) Expression level (qPCR) of CD45RABC and the constitutive exon9 using isoform specific oligonucleotides upon siDNMT3B and control siRNAs transfection; ( H ) Semi-quantitative PCR amplification of CD45 splicing isoforms in overexpressing-hnRNP-LL ICF1p2 sample upon siDNMT3B and control siRNAs transfection. * P -adj

    Article Snippet: Quantitative real time PCR Total RNA from B-LCLs was reverse-transcribed using iScript cDNA Synthesis kit (Bio-Rad San Diego, CA, USA).

    Techniques: Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Negative Control, Chromatin Immunoprecipitation, Expressing, Western Blot, CpG Methylation Assay, Methylation, Transfection, Amplification