quantitative real time pcr total rna  (Roche)


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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
    Quantitative Real Time Pcr Total Rna, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 85 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "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"

    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

    Journal: Cancer Science

    doi: 10.1111/cas.13747

    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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

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

    2) Product Images from "Inhibition of hepatic scavenger receptor-class B type I by RNA interference decreases atherosclerosis in rabbits"

    Article Title: Inhibition of hepatic scavenger receptor-class B type I by RNA interference decreases atherosclerosis in rabbits

    Journal: Atherosclerosis

    doi: 10.1016/j.atherosclerosis.2012.03.012

    Rabbits on cholesterol-rich diet were treated with SR-BI specific small hairpin RNA (grey circles) and respective scrambled controls (black circles) for 8 weeks. Total cholesterol levels were analyzed weekly (A). The insert shows the same curves in linear scale. Cholesterol measurements of lipoproteins fractions isolated from hyperlipidemic plasma samples by stepwise ultracentrifugation are shown in (B). Down-regulation of hepatic SR-BI expression on RNA level was shown by real-time PCR (C). Two representative aortas with Sudan IV stained lipid depositions in the intima (D), and the relative areas of atherosclerotic plaques in whole thoracic aortas and in corresponding aortic arches are presented (E). siRNA = small interfering RNA.
    Figure Legend Snippet: Rabbits on cholesterol-rich diet were treated with SR-BI specific small hairpin RNA (grey circles) and respective scrambled controls (black circles) for 8 weeks. Total cholesterol levels were analyzed weekly (A). The insert shows the same curves in linear scale. Cholesterol measurements of lipoproteins fractions isolated from hyperlipidemic plasma samples by stepwise ultracentrifugation are shown in (B). Down-regulation of hepatic SR-BI expression on RNA level was shown by real-time PCR (C). Two representative aortas with Sudan IV stained lipid depositions in the intima (D), and the relative areas of atherosclerotic plaques in whole thoracic aortas and in corresponding aortic arches are presented (E). siRNA = small interfering RNA.

    Techniques Used: Isolation, Expressing, Real-time Polymerase Chain Reaction, Staining, Small Interfering RNA

    3) Product Images from "CXCL9 induces chemotaxis, chemorepulsion and endothelial barrier disruption through CXCR3-mediated activation of melanoma cells"

    Article Title: CXCL9 induces chemotaxis, chemorepulsion and endothelial barrier disruption through CXCR3-mediated activation of melanoma cells

    Journal: British Journal of Cancer

    doi: 10.1038/sj.bjc.6606056

    Expression of CXCR3 ligands, CXCL9 and CXCL10, in human tumour endothelial cells. ( A ) Immunolocalisation of CXCL9 in human skin and melanoma metastases. Cryosections of normal human skin and melanoma metastasis (lymph node) were stained with anti-CXCL9 antibody (Alexa-488), anti-CD144 antibody (Alexa-568), and phalloidin-568 and viewed by confocal laser scanning microscopy; bar represents 50 μ m. Insert indicates tumour vessel in a higher magnification (dotted structure). ( B , C ) Quantitative Real-time PCR of CXCL9 and CXCL10. Blood ECs, LECs and TuECs were isolated from human skin and melanoma metastasis by cell sorting using anti-CD34, anti-CD144 and anti-podoplanin antibodies, total RNA was isolated, reverse transcribed and corresponding cDNA subjected to TaqMan PCR using commercial probes and primers for CXCL9 and CXCL10. All data shown are mean values and standard error of means from two independent PCR measurements, and have been normalised to the internal control gene B2M (N, normal skin; T, malignant melanoma).
    Figure Legend Snippet: Expression of CXCR3 ligands, CXCL9 and CXCL10, in human tumour endothelial cells. ( A ) Immunolocalisation of CXCL9 in human skin and melanoma metastases. Cryosections of normal human skin and melanoma metastasis (lymph node) were stained with anti-CXCL9 antibody (Alexa-488), anti-CD144 antibody (Alexa-568), and phalloidin-568 and viewed by confocal laser scanning microscopy; bar represents 50 μ m. Insert indicates tumour vessel in a higher magnification (dotted structure). ( B , C ) Quantitative Real-time PCR of CXCL9 and CXCL10. Blood ECs, LECs and TuECs were isolated from human skin and melanoma metastasis by cell sorting using anti-CD34, anti-CD144 and anti-podoplanin antibodies, total RNA was isolated, reverse transcribed and corresponding cDNA subjected to TaqMan PCR using commercial probes and primers for CXCL9 and CXCL10. All data shown are mean values and standard error of means from two independent PCR measurements, and have been normalised to the internal control gene B2M (N, normal skin; T, malignant melanoma).

    Techniques Used: Expressing, Staining, Confocal Laser Scanning Microscopy, Real-time Polymerase Chain Reaction, Isolation, FACS, Polymerase Chain Reaction

    4) Product Images from "Altered expression pattern of circular RNAs in primary and metastatic sites of epithelial ovarian carcinoma"

    Article Title: Altered expression pattern of circular RNAs in primary and metastatic sites of epithelial ovarian carcinoma

    Journal: Oncotarget

    doi: 10.18632/oncotarget.8917

    A. Divergent primers become properly inward facing and identify backspliced junction for circRNAs. Two sets of outward facing primers (a b) with respect to genomic sequence were designed to amplify the backsplice junction sequence for circRNA candidates from ARHGAP5, SPECC1 and NFATC3 genes and each produced an expected size band in the qPCR assay ( Supplementary File S1 ). B. Sanger sequencing confirms head-to-tail splicing for NFATC3. RNA samples were reverse transcribed with thermo-stable reverse transcriptase and a primer complementary to the coding RNA strand. PCR on the cDNA produced with primers 1 and 2 resulted in large amounts of product that could be Sanger sequenced. Primers 1 to 4 were used in sequencing, with coverage from each primer indicated in green. Expansion of the sequence provided by primer 3 shows complete coverage of the non-canonical junction. C. circRNA candidates are enriched after the RNase R digestion. Total RNA from OVCAR3 and SKOV3 cell lines was treated with RNAse R to test the sensitivity of the identified circRNA candidates to exoribonuclease digestion. Boxplots represent FPKM (Fragments per kilobase of exons per million mapped fragments) fold change for RNAse R treated vs. untreated samples and shows a significant enrichment of both junctional and supported circRNAs (n=7903) compared to mRNA (n=2785) in the RNAse R digested samples. Only genes that produced at least one detectable circular isoform were considered for this analysis. D. qPCR confirms RNAse R enrichment for circular junctions. Total RNA from OVCAR3 cell line was first treated with DNase1 to avoid any DNA contamination and then part of it was subjected to the RNase R digestion followed by RT-PCR for each sample. The four putative circular RNAs show enrichment for the backsplice junction compared to the linear RNA from beta-actin used as a negative control. E. Candidate circRNAs are devoid of Poly-A tails. The four candidate circRNAs were also tested for the presence /absence of a ploy-A tail by using either oligo (dT) or random hexamers to amplify the total RNA followed by a qPCR assay with primers specific for backsplice junctions of candidate circRNAs and linear RNA of b-actin and GAPDH genes. While both oligo (dT) and random hexamers could amplify the RNA from beta-actin and GAPDH, only random hexamers showed detectable PCR products for the four tested candidate circRNAs. The red rectangles show absence of any PCR bands for the backsplice junctions.
    Figure Legend Snippet: A. Divergent primers become properly inward facing and identify backspliced junction for circRNAs. Two sets of outward facing primers (a b) with respect to genomic sequence were designed to amplify the backsplice junction sequence for circRNA candidates from ARHGAP5, SPECC1 and NFATC3 genes and each produced an expected size band in the qPCR assay ( Supplementary File S1 ). B. Sanger sequencing confirms head-to-tail splicing for NFATC3. RNA samples were reverse transcribed with thermo-stable reverse transcriptase and a primer complementary to the coding RNA strand. PCR on the cDNA produced with primers 1 and 2 resulted in large amounts of product that could be Sanger sequenced. Primers 1 to 4 were used in sequencing, with coverage from each primer indicated in green. Expansion of the sequence provided by primer 3 shows complete coverage of the non-canonical junction. C. circRNA candidates are enriched after the RNase R digestion. Total RNA from OVCAR3 and SKOV3 cell lines was treated with RNAse R to test the sensitivity of the identified circRNA candidates to exoribonuclease digestion. Boxplots represent FPKM (Fragments per kilobase of exons per million mapped fragments) fold change for RNAse R treated vs. untreated samples and shows a significant enrichment of both junctional and supported circRNAs (n=7903) compared to mRNA (n=2785) in the RNAse R digested samples. Only genes that produced at least one detectable circular isoform were considered for this analysis. D. qPCR confirms RNAse R enrichment for circular junctions. Total RNA from OVCAR3 cell line was first treated with DNase1 to avoid any DNA contamination and then part of it was subjected to the RNase R digestion followed by RT-PCR for each sample. The four putative circular RNAs show enrichment for the backsplice junction compared to the linear RNA from beta-actin used as a negative control. E. Candidate circRNAs are devoid of Poly-A tails. The four candidate circRNAs were also tested for the presence /absence of a ploy-A tail by using either oligo (dT) or random hexamers to amplify the total RNA followed by a qPCR assay with primers specific for backsplice junctions of candidate circRNAs and linear RNA of b-actin and GAPDH genes. While both oligo (dT) and random hexamers could amplify the RNA from beta-actin and GAPDH, only random hexamers showed detectable PCR products for the four tested candidate circRNAs. The red rectangles show absence of any PCR bands for the backsplice junctions.

    Techniques Used: Sequencing, Produced, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Negative Control

    5) Product Images from "Nucleus accumbens-associated protein-1 promotes glycolysis and survival of hypoxic tumor cells via the HDAC4-HIF-1α axis"

    Article Title: Nucleus accumbens-associated protein-1 promotes glycolysis and survival of hypoxic tumor cells via the HDAC4-HIF-1α axis

    Journal: Oncogene

    doi: 10.1038/onc.2017.51

    Expression of NAC1 promotes glycolysis in hypoxic tumor cells. ( a ) HeLa cells were transfected with a non-targeting RNA (siNT) or NAC1-targeted siRNA, and exposed to hypoxia (1% O 2 ) for 24 h. Metabolic intermediates were measured using liquid chromatography-mass spectrometry (LC-MS), and normalized to cell numbers. ( b ) HeLa or SKOV3 cells were transfected with a siNT or NAC1 siRNA, and then incubated under normoxia (20% O 2 ) or hypoxia (1% O 2 ) for 24 h. Lactate and glucose in the culture medium, and cellular ATP, were measured and normalized to cell numbers. ( c ) ES-2 cells were transfected with an empty vector or V5-NAC1 expression vector. Twelve hours later, cells were cultured under hypoxia for 24 h. Lactate and glucose in the culture medium, and cellular ATP, were measured and normalized to cell numbers. ( d ) HeLa cells were transfected with a siNT or NAC1 siRNA, followed by incubation under normoxia or hypoxia for 24 h. mRNAs of lactate dehydrogenase-A (LDHA) and GLUT1 were determined by quantitative reverse transcriptase (RT)–PCR and plotted after normalization. LDHA and GLUT1 protein were examined by western blot, with β-actin as a loading control. Bars are mean ±s.d. ( n =3). ( e ) Glucose uptake and tumor growth in mice inoculated with Hela cells expressing a control shRNA or NAC1-targeted shRNA. 18 F-FDG uptake was analyzed by Micro PET. White squares point to tumors. Data shown are mean±s.d. ( n =5).
    Figure Legend Snippet: Expression of NAC1 promotes glycolysis in hypoxic tumor cells. ( a ) HeLa cells were transfected with a non-targeting RNA (siNT) or NAC1-targeted siRNA, and exposed to hypoxia (1% O 2 ) for 24 h. Metabolic intermediates were measured using liquid chromatography-mass spectrometry (LC-MS), and normalized to cell numbers. ( b ) HeLa or SKOV3 cells were transfected with a siNT or NAC1 siRNA, and then incubated under normoxia (20% O 2 ) or hypoxia (1% O 2 ) for 24 h. Lactate and glucose in the culture medium, and cellular ATP, were measured and normalized to cell numbers. ( c ) ES-2 cells were transfected with an empty vector or V5-NAC1 expression vector. Twelve hours later, cells were cultured under hypoxia for 24 h. Lactate and glucose in the culture medium, and cellular ATP, were measured and normalized to cell numbers. ( d ) HeLa cells were transfected with a siNT or NAC1 siRNA, followed by incubation under normoxia or hypoxia for 24 h. mRNAs of lactate dehydrogenase-A (LDHA) and GLUT1 were determined by quantitative reverse transcriptase (RT)–PCR and plotted after normalization. LDHA and GLUT1 protein were examined by western blot, with β-actin as a loading control. Bars are mean ±s.d. ( n =3). ( e ) Glucose uptake and tumor growth in mice inoculated with Hela cells expressing a control shRNA or NAC1-targeted shRNA. 18 F-FDG uptake was analyzed by Micro PET. White squares point to tumors. Data shown are mean±s.d. ( n =5).

    Techniques Used: Expressing, Transfection, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Incubation, Plasmid Preparation, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Western Blot, Mouse Assay, shRNA, Micro-PET

    6) Product Images from "Resveratrol Prevents EBV Transformation and Inhibits the Outgrowth of EBV-Immortalized Human B Cells"

    Article Title: Resveratrol Prevents EBV Transformation and Inhibits the Outgrowth of EBV-Immortalized Human B Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0051306

    Resveratrol downregulates LMP1 expression in EBV-infected B cells. (A) EBV-infected B cells were cultured for up to 96 hours with the indicated concentrations of resveratrol. Cellular RNA was extracted and the levels of LMP1 transcripts were determined using qRT-PCR. Error bars represent means±SEM of three independent experiments (B) B cells were infected with EBV for 72 hours and then cultured in the presence or absence of resveratrol for another 48 hours, after which the expression of LMP1 proteins was assessed using Western blotting. (C) Two EBV-immortalized LCLs were cultured with or without resveratrol for 48 hours and the levels of LMP1 transcripts were assessed using RT-PCR. Error bars represent means±SEM of three independent experiments (D) Whole-cell proteins from LCLs that were treated as in panel C were subjected to Western blotting and probed with anti-LMP1 antibodies. A representative figure of three independent experiments is shown. LCLs were treated as in panel C and the whole-cell protein extracts were analyzed using Western blotting with antibodies specific for EBNA1 (E) and EBNA-2 (F). The figures shown are representative results of three independent experiments.
    Figure Legend Snippet: Resveratrol downregulates LMP1 expression in EBV-infected B cells. (A) EBV-infected B cells were cultured for up to 96 hours with the indicated concentrations of resveratrol. Cellular RNA was extracted and the levels of LMP1 transcripts were determined using qRT-PCR. Error bars represent means±SEM of three independent experiments (B) B cells were infected with EBV for 72 hours and then cultured in the presence or absence of resveratrol for another 48 hours, after which the expression of LMP1 proteins was assessed using Western blotting. (C) Two EBV-immortalized LCLs were cultured with or without resveratrol for 48 hours and the levels of LMP1 transcripts were assessed using RT-PCR. Error bars represent means±SEM of three independent experiments (D) Whole-cell proteins from LCLs that were treated as in panel C were subjected to Western blotting and probed with anti-LMP1 antibodies. A representative figure of three independent experiments is shown. LCLs were treated as in panel C and the whole-cell protein extracts were analyzed using Western blotting with antibodies specific for EBNA1 (E) and EBNA-2 (F). The figures shown are representative results of three independent experiments.

    Techniques Used: Expressing, Infection, Cell Culture, Quantitative RT-PCR, Western Blot, Reverse Transcription Polymerase Chain Reaction

    Resveratrol inhibits the expression of viral-induced anti-apoptotic genes in EBV-infected B cells. (A) Primary B cells were infected with EBV and cultured for the indicated times in the absence or presence of resveratrol (50 µM). The level of BHRF1 transcripts normalized to U6B RNA, were measured by qRT-PCR using a lytic infected LCL as a reference control sample. Each data point shown represents the mean±SEM of three independent experiments (B). B cells were infected with EBV and cultured for 72 hours. They were then collected and treated for another 24 hours with or without resveratrol and the BHRF1 transcripts were measured by qRT-PCR (C) EBV infected B cells were cultured for the indicated times in the presence or absence of resveratrol (50 µM) and their levels of miR155 were measured by qRT-PCR (D) EBV-immortalized LCLs (1×10 6 ) were treated with resveratrol for 24 hrs and the expression of miR-155 and miR-34a were determined by qRT-PCR. The bars in figures B and D represent the means±SEM of three independent experiments.
    Figure Legend Snippet: Resveratrol inhibits the expression of viral-induced anti-apoptotic genes in EBV-infected B cells. (A) Primary B cells were infected with EBV and cultured for the indicated times in the absence or presence of resveratrol (50 µM). The level of BHRF1 transcripts normalized to U6B RNA, were measured by qRT-PCR using a lytic infected LCL as a reference control sample. Each data point shown represents the mean±SEM of three independent experiments (B). B cells were infected with EBV and cultured for 72 hours. They were then collected and treated for another 24 hours with or without resveratrol and the BHRF1 transcripts were measured by qRT-PCR (C) EBV infected B cells were cultured for the indicated times in the presence or absence of resveratrol (50 µM) and their levels of miR155 were measured by qRT-PCR (D) EBV-immortalized LCLs (1×10 6 ) were treated with resveratrol for 24 hrs and the expression of miR-155 and miR-34a were determined by qRT-PCR. The bars in figures B and D represent the means±SEM of three independent experiments.

    Techniques Used: Expressing, Infection, Cell Culture, Quantitative RT-PCR

    7) Product Images from "Coregulation of FANCA and BRCA1 in human cells"

    Article Title: Coregulation of FANCA and BRCA1 in human cells

    Journal: SpringerPlus

    doi: 10.1186/2193-1801-3-381

    Differential cell cycle regulation of FA genes in human T98G cells. (a-left panel) FACS analysis of synchronized cells, the 0, 8, 12, 16, 18, 20, 22, 24, 28, and 32 hr time points are shown; S phase is most pronounced between 18–22 hrs. Data of FACS represents one representative synchronization (a-right panel) Control experiment: The mRNA levels of CCNE2 during the cell cycle are shown. Quantitative RT-PCR was performed on total RNA samples from different time points and mean fold changes (MFC) were calculated relative to time point zero. Data represents the average mean fold change of 2–3 independent synchronization each in duplicate qPCR measurement (except for time points: 28 and 32) (b-g) Relative gene expression during the cell cycle for genes encoding in the FA/BRCA-pathway.
    Figure Legend Snippet: Differential cell cycle regulation of FA genes in human T98G cells. (a-left panel) FACS analysis of synchronized cells, the 0, 8, 12, 16, 18, 20, 22, 24, 28, and 32 hr time points are shown; S phase is most pronounced between 18–22 hrs. Data of FACS represents one representative synchronization (a-right panel) Control experiment: The mRNA levels of CCNE2 during the cell cycle are shown. Quantitative RT-PCR was performed on total RNA samples from different time points and mean fold changes (MFC) were calculated relative to time point zero. Data represents the average mean fold change of 2–3 independent synchronization each in duplicate qPCR measurement (except for time points: 28 and 32) (b-g) Relative gene expression during the cell cycle for genes encoding in the FA/BRCA-pathway.

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

    8) Product Images from "RANTES mediates kidney ischemia reperfusion injury through a possible role of HIF-1α and LncRNA PRINS"

    Article Title: RANTES mediates kidney ischemia reperfusion injury through a possible role of HIF-1α and LncRNA PRINS

    Journal: Scientific Reports

    doi: 10.1038/srep18424

    LncRNA-PRINS and RANTES interacts under hypoxia. ( a ) Deregulated LncRNA in hypoxia/normoxia condition for 24 hours. ( b ) Deregulated LncRNA in HK-2 sh HIF-1α and HK- 2 cells under hypoxia 24 hours. Briefly, cells were exposed to normoxia/hypoxia for 24 hours, after treatment RNA was isolated and Disease Related LncRNA array was performed. Each bar represents expression of Lnc-RNA relative to internal control. The results show 3 independent experiments performed in triplicates ( c ) Differentially expressed LncRNA in HIF-1α dependent manner. ( d ) Time course study on RANTES mRNA expression by Q-PCR. ( e ) Q-PCR analysis of RANTES and LncRNA-PRINS under normoxia and hypoxia 3 and 6 hour. Data are expressed as relative levels to RPLP0. The data are shown as the mean ± SD of three independent experiments performed in triplicates. ( f ) Luciferase reporter assay: The LncRNA-PRINS luciferase construct vector was transfected into WT-HK-2 cells for 24 hour and incubated under hypoxia and normoxia conditions for 3 and 6 hour respectively. Results were expressed in luciferase activity normalized to Renilla. The data are shown as the mean ± SD of three independent experiments performed in triplicates.
    Figure Legend Snippet: LncRNA-PRINS and RANTES interacts under hypoxia. ( a ) Deregulated LncRNA in hypoxia/normoxia condition for 24 hours. ( b ) Deregulated LncRNA in HK-2 sh HIF-1α and HK- 2 cells under hypoxia 24 hours. Briefly, cells were exposed to normoxia/hypoxia for 24 hours, after treatment RNA was isolated and Disease Related LncRNA array was performed. Each bar represents expression of Lnc-RNA relative to internal control. The results show 3 independent experiments performed in triplicates ( c ) Differentially expressed LncRNA in HIF-1α dependent manner. ( d ) Time course study on RANTES mRNA expression by Q-PCR. ( e ) Q-PCR analysis of RANTES and LncRNA-PRINS under normoxia and hypoxia 3 and 6 hour. Data are expressed as relative levels to RPLP0. The data are shown as the mean ± SD of three independent experiments performed in triplicates. ( f ) Luciferase reporter assay: The LncRNA-PRINS luciferase construct vector was transfected into WT-HK-2 cells for 24 hour and incubated under hypoxia and normoxia conditions for 3 and 6 hour respectively. Results were expressed in luciferase activity normalized to Renilla. The data are shown as the mean ± SD of three independent experiments performed in triplicates.

    Techniques Used: Isolation, Expressing, Polymerase Chain Reaction, Luciferase, Reporter Assay, Construct, Plasmid Preparation, Transfection, Incubation, Activity Assay

    9) Product Images from "The Varicella-Zoster Virus ORF47 Kinase Interferes with Host Innate Immune Response by Inhibiting the Activation of IRF3"

    Article Title: The Varicella-Zoster Virus ORF47 Kinase Interferes with Host Innate Immune Response by Inhibiting the Activation of IRF3

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0016870

    ORF47p is not responsible for the inhibition of NF-κB pathway during VZV infection. (A) Raw 264.7 macrophages were stimulated with LPS for 60 min. HEK-293 cells stably expressing TLR3 were stimulated or not with Poly (I:C) for 90 min. HEK-293 cells were mock-infected or infected with either the WT VZV or the mutant VZV ROka47S during the indicated periods of time. Various times post-infection, cells were collected and nuclear extracts were performed. 5 µg of nuclear proteins were used to study the NF-κB binding activity by EMSA using a radiolabeled probe carrying the NF-κB consensus sequence of HIV LTR promoter. (B) HEK-293 cells stably expressing TLR3 were stimulated or not with TNFα for 2 hours. Cells were mock-infected or infected with either the WT VZV or the mutant VZV ROka47S for 8 hours. Total RNA were harvested and subjected to a Reverse Transcription reaction. cDNA were subjected to a quantitative Real Time PCR in presence of SYBR Green and primers directed to iκbα . RT-PCR was normalized using the 18S RNA expression level. Error bars indicate the standard deviation of the mean.
    Figure Legend Snippet: ORF47p is not responsible for the inhibition of NF-κB pathway during VZV infection. (A) Raw 264.7 macrophages were stimulated with LPS for 60 min. HEK-293 cells stably expressing TLR3 were stimulated or not with Poly (I:C) for 90 min. HEK-293 cells were mock-infected or infected with either the WT VZV or the mutant VZV ROka47S during the indicated periods of time. Various times post-infection, cells were collected and nuclear extracts were performed. 5 µg of nuclear proteins were used to study the NF-κB binding activity by EMSA using a radiolabeled probe carrying the NF-κB consensus sequence of HIV LTR promoter. (B) HEK-293 cells stably expressing TLR3 were stimulated or not with TNFα for 2 hours. Cells were mock-infected or infected with either the WT VZV or the mutant VZV ROka47S for 8 hours. Total RNA were harvested and subjected to a Reverse Transcription reaction. cDNA were subjected to a quantitative Real Time PCR in presence of SYBR Green and primers directed to iκbα . RT-PCR was normalized using the 18S RNA expression level. Error bars indicate the standard deviation of the mean.

    Techniques Used: Inhibition, Infection, Stable Transfection, Expressing, Mutagenesis, Binding Assay, Activity Assay, Sequencing, Real-time Polymerase Chain Reaction, SYBR Green Assay, Reverse Transcription Polymerase Chain Reaction, RNA Expression, Standard Deviation

    In absence of ORF47p expression, IRF3-dependent gene expression is increased. HEK-293 cells stably expressing TLR3 were mock-infected or infected with either the WT VZV or the mutant VZV ROka47S for 8 hours. Total RNA were harvested and subjected to a Reverse Transcription reaction. cDNA were subjected to a quantitative Real Time PCR in presence of SYBR Green. Primers directed to ifnβ (A) and isg15 (B) were used. RT-PCR was normalized using the 18S RNA expression level. Error bars indicate the standard deviation of the mean.
    Figure Legend Snippet: In absence of ORF47p expression, IRF3-dependent gene expression is increased. HEK-293 cells stably expressing TLR3 were mock-infected or infected with either the WT VZV or the mutant VZV ROka47S for 8 hours. Total RNA were harvested and subjected to a Reverse Transcription reaction. cDNA were subjected to a quantitative Real Time PCR in presence of SYBR Green. Primers directed to ifnβ (A) and isg15 (B) were used. RT-PCR was normalized using the 18S RNA expression level. Error bars indicate the standard deviation of the mean.

    Techniques Used: Expressing, Stable Transfection, Infection, Mutagenesis, Real-time Polymerase Chain Reaction, SYBR Green Assay, Reverse Transcription Polymerase Chain Reaction, RNA Expression, Standard Deviation

    10) Product Images from "High levels of CCL2 or CCL4 in the tumor microenvironment predict unfavorable survival in lung adenocarcinoma"

    Article Title: High levels of CCL2 or CCL4 in the tumor microenvironment predict unfavorable survival in lung adenocarcinoma

    Journal: Thoracic Cancer

    doi: 10.1111/1759-7714.12643

    CCL2 and CCL4 overexpression in lung adenocarcinoma (LUAD). ( a ) Western blot analysis showed that CCL2 and CCL4 were upregulated in LUAD but not in lung squamous cell carcinoma (LUSC). β‐Actin was used as a loading control. ( b ) Quantitative real‐time‐PCR showed high expression of CCL2 and CCL4 at RNA level in LUAD ( n = 19) compared to LUSC ( n = 28). *** P
    Figure Legend Snippet: CCL2 and CCL4 overexpression in lung adenocarcinoma (LUAD). ( a ) Western blot analysis showed that CCL2 and CCL4 were upregulated in LUAD but not in lung squamous cell carcinoma (LUSC). β‐Actin was used as a loading control. ( b ) Quantitative real‐time‐PCR showed high expression of CCL2 and CCL4 at RNA level in LUAD ( n = 19) compared to LUSC ( n = 28). *** P

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

    11) Product Images from "Anti-TNF certolizumab pegol induces antioxidant response in human monocytes via reverse signaling"

    Article Title: Anti-TNF certolizumab pegol induces antioxidant response in human monocytes via reverse signaling

    Journal: Arthritis Research & Therapy

    doi: 10.1186/s13075-016-0955-8

    Certolizumab pegol ( CZP ) induces heme oxygenase 1 ( HO-1 ) expression. Monocytes were incubated, or not, with CZP (5 μg/ml) for 4 h of culture. RNA was isolated and qRT-PCR was performed to quantify HO-1 mRNA. Quantification of five experiments. *Paired t test
    Figure Legend Snippet: Certolizumab pegol ( CZP ) induces heme oxygenase 1 ( HO-1 ) expression. Monocytes were incubated, or not, with CZP (5 μg/ml) for 4 h of culture. RNA was isolated and qRT-PCR was performed to quantify HO-1 mRNA. Quantification of five experiments. *Paired t test

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

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    Real-time Polymerase Chain Reaction:

    Article Title: CXCL9 induces chemotaxis, chemorepulsion and endothelial barrier disruption through CXCR3-mediated activation of melanoma cells
    Article Snippet: .. Quantitative real-time PCR Total RNA was extracted using the High Pure RNA Isolation Kit (Roche). .. RNA was reverse transcribed with the TaqMan 5′ nuclease RT–PCR assay (Applied Biosystems, Foster City, CA, USA).

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    Article Snippet: .. RNA isolation and real‐time PCR Total RNA was extracted from paraffin sections of 25 lymph nodes (11 reactive, 8 nodular follicular lymphoma, and 6 diffuse follicular lymphoma) using High Pure miRNA Isolation kit (Roche Diagnostics, Monza, Italy). .. The quantity and quality of the RNA was determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA USA).

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    Article Snippet: .. RNA ISOLATION AND REAL-TIME PCR Total RNA was extracted from the various treated cells using the High Pure RNA Isolation Kit (Roche, Germany). .. The RNA was reverse transcribed using a Verso cDNA kit (Thermo Scientific, Epson, Surrey, UK) using random primers.

    Article Title: Utilization of transposable element mPing as a novel genetic tool for modification of the stress response in rice
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    Article Title: Inhibition of Extracellular Calcium Influx Results in Enhanced IL-12 Production in LPS-Treated Murine Macrophages by Downregulation of the CaMKK β-AMPK-SIRT1 Signaling Pathway
    Article Snippet: .. Real-Time PCR Total RNA extracted from RAW 264.7 cells by a TRIzol reagent (Roche) was reversely transcribed into cDNA with a ReverTra Ace-α -RNA easy Kit (Toyobo, Japan). .. The cDNA templates were mixed with SYBR Green PCR Master Mix (Toyobo) and PCR primers for IL-12 p35, IL-12 p40, TNF-α , IL-6, CaMKKβ , AMPKα , and β -actin (sequences are listed in Table S1, in Supplementary Material available online at http://dx.doi.org/10.1155/2016/6152713 ).

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    Article Snippet: .. 2.4 RNA isolation, reverse transcription and quantitative real-time PCR Total RNA from SR-BI expressing HuH-7 cells and liver specimens was prepared using High Pure Isolation Kit (Roche, Mannheim, Germany) and reversely transcribed with Omniscript-RT Kit (Qiagen, Hilden, Germany). .. Primers and TaqMan probes for SR-BI, ATP-binding cassette A1, low-density lipoprotein receptor, and cholesterol 7α-hydroxylase were designed using Primer Express software (Applied Biosystems, Foster City, CA, USA).

    Isolation:

    Article Title: CXCL9 induces chemotaxis, chemorepulsion and endothelial barrier disruption through CXCR3-mediated activation of melanoma cells
    Article Snippet: .. Quantitative real-time PCR Total RNA was extracted using the High Pure RNA Isolation Kit (Roche). .. RNA was reverse transcribed with the TaqMan 5′ nuclease RT–PCR assay (Applied Biosystems, Foster City, CA, USA).

    Article Title: Expression of a urokinase‐type plasminogen activator during tumor growth leads to angiogenesis via galanin activation in tumor‐bearing mice
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    Article Title: Inhibition of hepatic scavenger receptor-class B type I by RNA interference decreases atherosclerosis in rabbits
    Article Snippet: .. 2.4 RNA isolation, reverse transcription and quantitative real-time PCR Total RNA from SR-BI expressing HuH-7 cells and liver specimens was prepared using High Pure Isolation Kit (Roche, Mannheim, Germany) and reversely transcribed with Omniscript-RT Kit (Qiagen, Hilden, Germany). .. Primers and TaqMan probes for SR-BI, ATP-binding cassette A1, low-density lipoprotein receptor, and cholesterol 7α-hydroxylase were designed using Primer Express software (Applied Biosystems, Foster City, CA, USA).

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    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. .. The first line of cDNA was synthesized using a Reverse Transcription Kit (Takara, Dalian, China).

    Cell Culture:

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    Expressing:

    Article Title: Inhibition of hepatic scavenger receptor-class B type I by RNA interference decreases atherosclerosis in rabbits
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    Roche rna
    Chronic hypoxia‐induced slug activates expression of ephrin‐B1 through E‐box motifs. A, Relative expression of ephrin‐B1 mRNA in non‐transfected (control), slug si <t>RNA</t> ‐transfected (siSlug) and control si RNA ‐transfected (siScr) LNC aP/ CH 6M cells. B, Relative expression of ephrin‐B1 mRNA in the LNC aP/N cells transfected with various amounts of the slug‐expression vector, pL enti6.3/V5‐ DEST ‐slug (0‐1000 ng). C, Schematic diagram of luciferase reporter constructs containing wild‐type human ephrin‐B1 promoter ( pGL ‐Basic‐ephrin‐B1 promoter WT ), E‐box1 (E1m) or E‐box2 (E2m) mutations, or double mutations (E1mE2m). Arrows, primer pairs flanking E‐box motifs used for chromatin immunoprecipitation (ChIP) analysis. D, Effect of slug overexpression on the wild‐type ephrin‐B1 promoter activity. LNC aP/N cells were transfected with 1000 ng pGL ‐Basic‐ephrin‐B1 promoter WT and various amounts of the slug‐expression vector, pL enti6.3/V5‐ DEST ‐slug (0‐1000 ng). Firefly luciferase activity was normalized to Renilla luciferase activity. E, Mutational analysis of the E‐box motifs in the ephrin‐B1 promoter. LNC aP/N cells were transfected with 1000 ng pL enti6.3/V5‐ DEST ‐slug, and 1000 ng pGL ‐Basic‐ephrin‐B1 promoter WT , E1m, E2m, or E1mE2m. F, Ch IP analysis of slug on the E‐box regions in the ephrin‐B1 promoter. Soluble chromatin extracted from LNC aP/ CH 6M cells was immunoprecipitated with anti‐slug and control IgG antibodies. Ch IP was analyzed on quantitative <t>PCR</t> using primers flanking the E‐box motifs, E‐box1 and E‐box2, shown in (C). Fold change compared with control IgG‐enriched DNA fragments was measured. Data given as mean ± SD . * P
    Rna, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 175 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche real time quantitative rt pcr qrt pcr total rna
    CD271 expression in pancreatic stellate cells (PSCs) decreases after long-term interactions with pancreatic cancer cells. (A) Real-time <t>qRT-PCR</t> analyses showed that the levels of CD271 mRNA expression in PSCs in coculture with pancreatic cancer cells start to increase on day 3 (p = 0.0249), are highest on day 4 (p
    Real Time Quantitative Rt Pcr Qrt Pcr Total Rna, supplied by Roche, used in various techniques. Bioz Stars score: 89/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    OL suppresses HFD-induced accumulation of M2-macrophages in the tumor tissues and adipose tissues surrounding the LNs ( A , B ) Tumor sections were stained with an antibody raised against F4/80 or MMR. (A) Representative immunofluorescence images are shown. (B) F4/80 and MMR-positive cells were quantified ( n = 5). ( C ) Total <t>RNA</t> was isolated from tumor tissues, and F4/80 and MMR gene expression levels were determined using real-time <t>RT-PCR</t> ( n = 9). ( D , E ) Sections of adipose tissues surrounding the LNs were stained with hematoxylin and eosin. Mature macrophages were identified using an F4/80 antibody (red) and adipocyte membranes a caveolin-1 antibody (green). Nuclei were counterstained with DAPI (blue). (D) Representative stained images are shown. (E) Mean diameters of adipocytes surrounding the LN were estimated ( n = 4). ( F ) F4/80 and MMR gene expression levels in fat tissues surrounding the LN, as determined using real-time RT-PCR ( n = 6). *Significantly different from the CD group, P
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    Roche quantitative real time pcr xs52 total rna
    S1P is generated by <t>XS52</t> cells and released to the extracellular environment. XS52 cells were cultivated over a time period of 6 h and S1P levels in the extracellular environment was detected (A). Quantitative real time <t>PCR</t> analysis of ABCB1, ABCC1, and ABCG2 of three different sets of cells was performed using HPRT1 and GADPH as reference genes (B). Cells were preincubated with Reversin 121 (10 µM), Fumitremorgin C (10 µM), and Probenecid (2.5 mM) for 6 h (C). Cells were transfected with siRNA against ABCC1 or control siRNA and silencing was detected by quantitative real time PCR (D). Then, cells were incubated with FITC-labeled dextran for 15 min. Fluorescence intensity of cells was analyzed by flow cytometry and relative endocytosis was calculated. Data are expressed as the mean ± SEM of results from at least three independent experiments. **P
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    Chronic hypoxia‐induced slug activates expression of ephrin‐B1 through E‐box motifs. A, Relative expression of ephrin‐B1 mRNA in non‐transfected (control), slug si RNA ‐transfected (siSlug) and control si RNA ‐transfected (siScr) LNC aP/ CH 6M cells. B, Relative expression of ephrin‐B1 mRNA in the LNC aP/N cells transfected with various amounts of the slug‐expression vector, pL enti6.3/V5‐ DEST ‐slug (0‐1000 ng). C, Schematic diagram of luciferase reporter constructs containing wild‐type human ephrin‐B1 promoter ( pGL ‐Basic‐ephrin‐B1 promoter WT ), E‐box1 (E1m) or E‐box2 (E2m) mutations, or double mutations (E1mE2m). Arrows, primer pairs flanking E‐box motifs used for chromatin immunoprecipitation (ChIP) analysis. D, Effect of slug overexpression on the wild‐type ephrin‐B1 promoter activity. LNC aP/N cells were transfected with 1000 ng pGL ‐Basic‐ephrin‐B1 promoter WT and various amounts of the slug‐expression vector, pL enti6.3/V5‐ DEST ‐slug (0‐1000 ng). Firefly luciferase activity was normalized to Renilla luciferase activity. E, Mutational analysis of the E‐box motifs in the ephrin‐B1 promoter. LNC aP/N cells were transfected with 1000 ng pL enti6.3/V5‐ DEST ‐slug, and 1000 ng pGL ‐Basic‐ephrin‐B1 promoter WT , E1m, E2m, or E1mE2m. F, Ch IP analysis of slug on the E‐box regions in the ephrin‐B1 promoter. Soluble chromatin extracted from LNC aP/ CH 6M cells was immunoprecipitated with anti‐slug and control IgG antibodies. Ch IP was analyzed on quantitative PCR using primers flanking the E‐box motifs, E‐box1 and E‐box2, shown in (C). Fold change compared with control IgG‐enriched DNA fragments was measured. Data given as mean ± SD . * P

    Journal: Cancer Science

    Article Title: Chronic hypoxia‐induced slug promotes invasive behavior of prostate cancer cells by activating expression of ephrin‐B1, et al. Chronic hypoxia‐induced slug promotes invasive behavior of prostate cancer cells by activating expression of ephrin‐B1

    doi: 10.1111/cas.13754

    Figure Lengend Snippet: Chronic hypoxia‐induced slug activates expression of ephrin‐B1 through E‐box motifs. A, Relative expression of ephrin‐B1 mRNA in non‐transfected (control), slug si RNA ‐transfected (siSlug) and control si RNA ‐transfected (siScr) LNC aP/ CH 6M cells. B, Relative expression of ephrin‐B1 mRNA in the LNC aP/N cells transfected with various amounts of the slug‐expression vector, pL enti6.3/V5‐ DEST ‐slug (0‐1000 ng). C, Schematic diagram of luciferase reporter constructs containing wild‐type human ephrin‐B1 promoter ( pGL ‐Basic‐ephrin‐B1 promoter WT ), E‐box1 (E1m) or E‐box2 (E2m) mutations, or double mutations (E1mE2m). Arrows, primer pairs flanking E‐box motifs used for chromatin immunoprecipitation (ChIP) analysis. D, Effect of slug overexpression on the wild‐type ephrin‐B1 promoter activity. LNC aP/N cells were transfected with 1000 ng pGL ‐Basic‐ephrin‐B1 promoter WT and various amounts of the slug‐expression vector, pL enti6.3/V5‐ DEST ‐slug (0‐1000 ng). Firefly luciferase activity was normalized to Renilla luciferase activity. E, Mutational analysis of the E‐box motifs in the ephrin‐B1 promoter. LNC aP/N cells were transfected with 1000 ng pL enti6.3/V5‐ DEST ‐slug, and 1000 ng pGL ‐Basic‐ephrin‐B1 promoter WT , E1m, E2m, or E1mE2m. F, Ch IP analysis of slug on the E‐box regions in the ephrin‐B1 promoter. Soluble chromatin extracted from LNC aP/ CH 6M cells was immunoprecipitated with anti‐slug and control IgG antibodies. Ch IP was analyzed on quantitative PCR using primers flanking the E‐box motifs, E‐box1 and E‐box2, shown in (C). Fold change compared with control IgG‐enriched DNA fragments was measured. Data given as mean ± SD . * P

    Article Snippet: 2.5 Real‐time quantitative RT‐PCR First‐strand cDNA was synthesized from the total RNA using ThermoScript RT‐PCR System (Roche, Indianapolis, IN, USA).

    Techniques: Expressing, Transfection, Plasmid Preparation, Luciferase, Construct, Chromatin Immunoprecipitation, Over Expression, Activity Assay, Immunoprecipitation, Real-time Polymerase Chain Reaction

    CD271 expression in pancreatic stellate cells (PSCs) decreases after long-term interactions with pancreatic cancer cells. (A) Real-time qRT-PCR analyses showed that the levels of CD271 mRNA expression in PSCs in coculture with pancreatic cancer cells start to increase on day 3 (p = 0.0249), are highest on day 4 (p

    Journal: PLoS ONE

    Article Title: CD271+ Subpopulation of Pancreatic Stellate Cells Correlates with Prognosis of Pancreatic Cancer and Is Regulated by Interaction with Cancer Cells

    doi: 10.1371/journal.pone.0052682

    Figure Lengend Snippet: CD271 expression in pancreatic stellate cells (PSCs) decreases after long-term interactions with pancreatic cancer cells. (A) Real-time qRT-PCR analyses showed that the levels of CD271 mRNA expression in PSCs in coculture with pancreatic cancer cells start to increase on day 3 (p = 0.0249), are highest on day 4 (p

    Article Snippet: Real-time quantitative RT-PCR (qRT-PCR) Total RNA was extracted from cultured cells using a High Pure RNA Isolation Kit (Roche Diagnostics, Mannheim, Germany) and DNase I (Roche Diagnostics).

    Techniques: Expressing, Quantitative RT-PCR

    OL suppresses HFD-induced accumulation of M2-macrophages in the tumor tissues and adipose tissues surrounding the LNs ( A , B ) Tumor sections were stained with an antibody raised against F4/80 or MMR. (A) Representative immunofluorescence images are shown. (B) F4/80 and MMR-positive cells were quantified ( n = 5). ( C ) Total RNA was isolated from tumor tissues, and F4/80 and MMR gene expression levels were determined using real-time RT-PCR ( n = 9). ( D , E ) Sections of adipose tissues surrounding the LNs were stained with hematoxylin and eosin. Mature macrophages were identified using an F4/80 antibody (red) and adipocyte membranes a caveolin-1 antibody (green). Nuclei were counterstained with DAPI (blue). (D) Representative stained images are shown. (E) Mean diameters of adipocytes surrounding the LN were estimated ( n = 4). ( F ) F4/80 and MMR gene expression levels in fat tissues surrounding the LN, as determined using real-time RT-PCR ( n = 6). *Significantly different from the CD group, P

    Journal: Oncotarget

    Article Title: Dietary oleuropein inhibits tumor angiogenesis and lymphangiogenesis in the B16F10 melanoma allograft model: a mechanism for the suppression of high-fat diet-induced solid tumor growth and lymph node metastasis

    doi: 10.18632/oncotarget.16757

    Figure Lengend Snippet: OL suppresses HFD-induced accumulation of M2-macrophages in the tumor tissues and adipose tissues surrounding the LNs ( A , B ) Tumor sections were stained with an antibody raised against F4/80 or MMR. (A) Representative immunofluorescence images are shown. (B) F4/80 and MMR-positive cells were quantified ( n = 5). ( C ) Total RNA was isolated from tumor tissues, and F4/80 and MMR gene expression levels were determined using real-time RT-PCR ( n = 9). ( D , E ) Sections of adipose tissues surrounding the LNs were stained with hematoxylin and eosin. Mature macrophages were identified using an F4/80 antibody (red) and adipocyte membranes a caveolin-1 antibody (green). Nuclei were counterstained with DAPI (blue). (D) Representative stained images are shown. (E) Mean diameters of adipocytes surrounding the LN were estimated ( n = 4). ( F ) F4/80 and MMR gene expression levels in fat tissues surrounding the LN, as determined using real-time RT-PCR ( n = 6). *Significantly different from the CD group, P

    Article Snippet: Real-time RT-PCR analysis Total RNA was isolated from tissues using TRIzol reagent (Roche, Indianapolis, IN, USA).

    Techniques: Staining, Immunofluorescence, Isolation, Expressing, Quantitative RT-PCR

    S1P is generated by XS52 cells and released to the extracellular environment. XS52 cells were cultivated over a time period of 6 h and S1P levels in the extracellular environment was detected (A). Quantitative real time PCR analysis of ABCB1, ABCC1, and ABCG2 of three different sets of cells was performed using HPRT1 and GADPH as reference genes (B). Cells were preincubated with Reversin 121 (10 µM), Fumitremorgin C (10 µM), and Probenecid (2.5 mM) for 6 h (C). Cells were transfected with siRNA against ABCC1 or control siRNA and silencing was detected by quantitative real time PCR (D). Then, cells were incubated with FITC-labeled dextran for 15 min. Fluorescence intensity of cells was analyzed by flow cytometry and relative endocytosis was calculated. Data are expressed as the mean ± SEM of results from at least three independent experiments. **P

    Journal: PLoS ONE

    Article Title: Sphingosine 1-Phosphate Modulates Antigen Capture by Murine Langerhans Cells via the S1P2 Receptor Subtype

    doi: 10.1371/journal.pone.0049427

    Figure Lengend Snippet: S1P is generated by XS52 cells and released to the extracellular environment. XS52 cells were cultivated over a time period of 6 h and S1P levels in the extracellular environment was detected (A). Quantitative real time PCR analysis of ABCB1, ABCC1, and ABCG2 of three different sets of cells was performed using HPRT1 and GADPH as reference genes (B). Cells were preincubated with Reversin 121 (10 µM), Fumitremorgin C (10 µM), and Probenecid (2.5 mM) for 6 h (C). Cells were transfected with siRNA against ABCC1 or control siRNA and silencing was detected by quantitative real time PCR (D). Then, cells were incubated with FITC-labeled dextran for 15 min. Fluorescence intensity of cells was analyzed by flow cytometry and relative endocytosis was calculated. Data are expressed as the mean ± SEM of results from at least three independent experiments. **P

    Article Snippet: Quantitative Real Time PCR XS52 total RNA was isolated using High Pure RNA Isolation Kit (Roche Applied Sciences, Mannheim, Germany) following quality check by spectroscopy.

    Techniques: Generated, Real-time Polymerase Chain Reaction, Transfection, Incubation, Labeling, Fluorescence, Flow Cytometry, Cytometry

    S1P inhibits macropinocytosis of FITC-labeled dextran via the S1P 2 receptor subtype. XS52 cells were transfected with siRNA against S1P 2 or control siRNA and silencing was detected by quantitative real time PCR (A). Transfected and control cells were incubated with FITC-labeled dextran in the presence or absence of S1P (5 µM) for 15 min and macropinocytosis was detected by flow cytometry. Relative endocytosis are expressed as the mean ± SEM of results from at least three independent experiments. **P

    Journal: PLoS ONE

    Article Title: Sphingosine 1-Phosphate Modulates Antigen Capture by Murine Langerhans Cells via the S1P2 Receptor Subtype

    doi: 10.1371/journal.pone.0049427

    Figure Lengend Snippet: S1P inhibits macropinocytosis of FITC-labeled dextran via the S1P 2 receptor subtype. XS52 cells were transfected with siRNA against S1P 2 or control siRNA and silencing was detected by quantitative real time PCR (A). Transfected and control cells were incubated with FITC-labeled dextran in the presence or absence of S1P (5 µM) for 15 min and macropinocytosis was detected by flow cytometry. Relative endocytosis are expressed as the mean ± SEM of results from at least three independent experiments. **P

    Article Snippet: Quantitative Real Time PCR XS52 total RNA was isolated using High Pure RNA Isolation Kit (Roche Applied Sciences, Mannheim, Germany) following quality check by spectroscopy.

    Techniques: Labeling, Transfection, Real-time Polymerase Chain Reaction, Incubation, Flow Cytometry, Cytometry