Structured Review

Roche qrt pcr analysis
High level of circulating SLURP1 is associated with better survival in operable PDAC patients ( A ) The measurement of pancreatic SLURP1 mRNA expression with <t>qRT-PCR</t> in 66 pancreatic samples revealed the lack of expression in non-malignant pancreatic tissues and most PDAC lesions. ( B ) The concentration of SLURP1 was measured in 106 human serum samples using a commercial EIA kit produced by CUSABIO. Multiple comparison testing revealed a lack of difference between the analyzed groups ( p = 0.643). ( C ) Circulating SLURP1 levels correlated with the grade of differentiation but not with any of the TNM parameters. ( D ) Dividing the PDAC patients with resected tumors into SLURP1 high/low groups according to the preoperative level of circulating SLURP1 for the Kaplan-Meier survival analysis revealed significantly longer survival of SLURP1 high patients (cut-off = 16 ng/ml; log-rank test p = 0.007), MS: median survival. ( E ) The concentration of SLURP1 was re-measured in 67 human serum samples using a commercial EIA kit produced by USCN and confirmed the lack of difference between the analyzed groups ( p = 0.951). ( F ) Nevertheless, the USCN-EIA recognized the CUSABIO standard but not vice versa, and the USCN values for SLURP1 were three-fold lower and lacked any correlation with the CUSABIO values. Additional information is presented in Table 1 . ( G ) Clustering analysis of the RNAseq data obtained for the PDAC specimens corresponding to the serum samples with low and high SLURP1 content.
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1) Product Images from "Endogenous CHRNA7-ligand SLURP1 as a potential tumor suppressor and anti-nicotinic factor in pancreatic cancer"

Article Title: Endogenous CHRNA7-ligand SLURP1 as a potential tumor suppressor and anti-nicotinic factor in pancreatic cancer

Journal: Oncotarget

doi: 10.18632/oncotarget.24312

High level of circulating SLURP1 is associated with better survival in operable PDAC patients ( A ) The measurement of pancreatic SLURP1 mRNA expression with qRT-PCR in 66 pancreatic samples revealed the lack of expression in non-malignant pancreatic tissues and most PDAC lesions. ( B ) The concentration of SLURP1 was measured in 106 human serum samples using a commercial EIA kit produced by CUSABIO. Multiple comparison testing revealed a lack of difference between the analyzed groups ( p = 0.643). ( C ) Circulating SLURP1 levels correlated with the grade of differentiation but not with any of the TNM parameters. ( D ) Dividing the PDAC patients with resected tumors into SLURP1 high/low groups according to the preoperative level of circulating SLURP1 for the Kaplan-Meier survival analysis revealed significantly longer survival of SLURP1 high patients (cut-off = 16 ng/ml; log-rank test p = 0.007), MS: median survival. ( E ) The concentration of SLURP1 was re-measured in 67 human serum samples using a commercial EIA kit produced by USCN and confirmed the lack of difference between the analyzed groups ( p = 0.951). ( F ) Nevertheless, the USCN-EIA recognized the CUSABIO standard but not vice versa, and the USCN values for SLURP1 were three-fold lower and lacked any correlation with the CUSABIO values. Additional information is presented in Table 1 . ( G ) Clustering analysis of the RNAseq data obtained for the PDAC specimens corresponding to the serum samples with low and high SLURP1 content.
Figure Legend Snippet: High level of circulating SLURP1 is associated with better survival in operable PDAC patients ( A ) The measurement of pancreatic SLURP1 mRNA expression with qRT-PCR in 66 pancreatic samples revealed the lack of expression in non-malignant pancreatic tissues and most PDAC lesions. ( B ) The concentration of SLURP1 was measured in 106 human serum samples using a commercial EIA kit produced by CUSABIO. Multiple comparison testing revealed a lack of difference between the analyzed groups ( p = 0.643). ( C ) Circulating SLURP1 levels correlated with the grade of differentiation but not with any of the TNM parameters. ( D ) Dividing the PDAC patients with resected tumors into SLURP1 high/low groups according to the preoperative level of circulating SLURP1 for the Kaplan-Meier survival analysis revealed significantly longer survival of SLURP1 high patients (cut-off = 16 ng/ml; log-rank test p = 0.007), MS: median survival. ( E ) The concentration of SLURP1 was re-measured in 67 human serum samples using a commercial EIA kit produced by USCN and confirmed the lack of difference between the analyzed groups ( p = 0.951). ( F ) Nevertheless, the USCN-EIA recognized the CUSABIO standard but not vice versa, and the USCN values for SLURP1 were three-fold lower and lacked any correlation with the CUSABIO values. Additional information is presented in Table 1 . ( G ) Clustering analysis of the RNAseq data obtained for the PDAC specimens corresponding to the serum samples with low and high SLURP1 content.

Techniques Used: Expressing, Quantitative RT-PCR, Concentration Assay, Enzyme-linked Immunosorbent Assay, Produced, Mass Spectrometry

Preservation of CHRNA7 expression in the pancreas is associated with better prognosis for operable PDAC patients ( A ) CHRNA7 mRNA expression was determined by qRT-PCR in 66 pancreatic samples obtained from the organ donors and patients with chronic pancreatitis (CP) or pancreatic adenocarcinoma (PDAC). The groups were compared using the Kruskal-Wallis test ( p = 0.004) with Dunn’s procedure, which established significant down-regulation of CHRNA7 in PDAC patients ( p
Figure Legend Snippet: Preservation of CHRNA7 expression in the pancreas is associated with better prognosis for operable PDAC patients ( A ) CHRNA7 mRNA expression was determined by qRT-PCR in 66 pancreatic samples obtained from the organ donors and patients with chronic pancreatitis (CP) or pancreatic adenocarcinoma (PDAC). The groups were compared using the Kruskal-Wallis test ( p = 0.004) with Dunn’s procedure, which established significant down-regulation of CHRNA7 in PDAC patients ( p

Techniques Used: Preserving, Expressing, Quantitative RT-PCR

CHRNA7 is a major binding site for SLURP1 on PDAC cells ( A ) Simultaneous FACS and qRT-PCR analyses revealed CHRNA7-positivity of all eight tested PDAC cell lines, whereby membranous CHRNA7-staining showed significant inter-experimental variability and lack of correlation with RNA levels. The graph summarizes the data of two independent experiments shown below and measuring CHRNA7 mRNA (qRT-PCR; log10-transformed number of transcripts/10k PPIB) and surface protein expression (log10-transformed mean fluorescence intensity/MFI values) in unrelated passages of each cell line. ( B ) Individual FACS histograms illustrating intra- and inter-experimental variability are shown as an overlap of anti-CHRNA7-IgG (gray tinted area) and rabbit IgG (bold black line; isotype control) profiles; the difference in their geometric MFIs was used to calculate final MFI value for each culture. ( C ) qRT-PCR data demonstrating the inter-experimental stability of CHRNA7 mRNA expression also revealed the existence of the two groups, whereby CHRNA7 high -group but not CHRNA7 neg/low -group showed RNA-agreeable, stable pattern of protein expression. ( D – F ) The siRNA-based depletion of CHRNA7 confirmed the specificity of the reagents such as primers (qRT-PCR, D) and antibodies (Western blot, E, and FACS, F). As illustrated for COLO357 cultures, transfection with three commercially available CHRNA7-specific siRNA sets (si#1, si#2, si#3) eliminated over 75% of CHRNA7 mRNA and protein compared to the transfection of cells with negative control siRNA set (neg.si) or left untreated (control). ( G ) siRNA-based depletion of CHRNA7 indicated the preferential binding of SLURP1 to this receptor on PDAC cells. For comparison, FITC-conjugated SLURP1 was added to COLO357 and PANC-1 cells transfected with three different CHRNA7-siRNA sets (‘CHRNA7-depletion’ group depicted by grey-, green- and rose-colored profiles for si#1, si#2 and si#3, respectively) and to cells remaining intact or transfected with negative siRNA (‘controls’ group depicted by blue and lilac-colored profiles).
Figure Legend Snippet: CHRNA7 is a major binding site for SLURP1 on PDAC cells ( A ) Simultaneous FACS and qRT-PCR analyses revealed CHRNA7-positivity of all eight tested PDAC cell lines, whereby membranous CHRNA7-staining showed significant inter-experimental variability and lack of correlation with RNA levels. The graph summarizes the data of two independent experiments shown below and measuring CHRNA7 mRNA (qRT-PCR; log10-transformed number of transcripts/10k PPIB) and surface protein expression (log10-transformed mean fluorescence intensity/MFI values) in unrelated passages of each cell line. ( B ) Individual FACS histograms illustrating intra- and inter-experimental variability are shown as an overlap of anti-CHRNA7-IgG (gray tinted area) and rabbit IgG (bold black line; isotype control) profiles; the difference in their geometric MFIs was used to calculate final MFI value for each culture. ( C ) qRT-PCR data demonstrating the inter-experimental stability of CHRNA7 mRNA expression also revealed the existence of the two groups, whereby CHRNA7 high -group but not CHRNA7 neg/low -group showed RNA-agreeable, stable pattern of protein expression. ( D – F ) The siRNA-based depletion of CHRNA7 confirmed the specificity of the reagents such as primers (qRT-PCR, D) and antibodies (Western blot, E, and FACS, F). As illustrated for COLO357 cultures, transfection with three commercially available CHRNA7-specific siRNA sets (si#1, si#2, si#3) eliminated over 75% of CHRNA7 mRNA and protein compared to the transfection of cells with negative control siRNA set (neg.si) or left untreated (control). ( G ) siRNA-based depletion of CHRNA7 indicated the preferential binding of SLURP1 to this receptor on PDAC cells. For comparison, FITC-conjugated SLURP1 was added to COLO357 and PANC-1 cells transfected with three different CHRNA7-siRNA sets (‘CHRNA7-depletion’ group depicted by grey-, green- and rose-colored profiles for si#1, si#2 and si#3, respectively) and to cells remaining intact or transfected with negative siRNA (‘controls’ group depicted by blue and lilac-colored profiles).

Techniques Used: Binding Assay, FACS, Quantitative RT-PCR, Staining, Transformation Assay, Expressing, Fluorescence, Western Blot, Transfection, Negative Control

2) Product Images from "The novel lncRNA lnc-NR2F1 is pro-neurogenic and mutated in human neurodevelopmental disorders"

Article Title: The novel lncRNA lnc-NR2F1 is pro-neurogenic and mutated in human neurodevelopmental disorders

Journal: eLife

doi: 10.7554/eLife.41770

Characterization of the epistasis relationship between mouse Nr2f1 and lnc-Nr2f1 . ( A ) CRISPR knock out strategy to generate Nr2f1 knockout (Homo) and heterozygous null lines (Het) from the control mES cells (Ctrl). ( B ) qRT-PCR results for lnc-Nr2f1 and Nr2f1 in the Ctrl, Nr2f1 heterozygous null and Nr2f1 knock out day 4 Ngn2 mES-iN. (n = 4 for Ctrl and Homo, n = 6 for Het; n.s. denotes not significant by two tailed t test) ( C ) Western blot showing the level of NR2F1 for individual clones of Ctrl, Het and Homo for day 4 Ngn2 mES-iN. ( D ) Neurite length measurement of the Ngn2 day 3 mES iN cells generated from the Nr2f1 Ctrl, Het or Homo lines. (n = 4 for Ctrl and Homo, n = 6 for Het) (n.s. indicates p
Figure Legend Snippet: Characterization of the epistasis relationship between mouse Nr2f1 and lnc-Nr2f1 . ( A ) CRISPR knock out strategy to generate Nr2f1 knockout (Homo) and heterozygous null lines (Het) from the control mES cells (Ctrl). ( B ) qRT-PCR results for lnc-Nr2f1 and Nr2f1 in the Ctrl, Nr2f1 heterozygous null and Nr2f1 knock out day 4 Ngn2 mES-iN. (n = 4 for Ctrl and Homo, n = 6 for Het; n.s. denotes not significant by two tailed t test) ( C ) Western blot showing the level of NR2F1 for individual clones of Ctrl, Het and Homo for day 4 Ngn2 mES-iN. ( D ) Neurite length measurement of the Ngn2 day 3 mES iN cells generated from the Nr2f1 Ctrl, Het or Homo lines. (n = 4 for Ctrl and Homo, n = 6 for Het) (n.s. indicates p

Techniques Used: CRISPR, Knock-Out, Quantitative RT-PCR, Two Tailed Test, Western Blot, Clone Assay, Generated

QRT-PCR validation of candidate lncRNAs expression. ( A ) Expression detection of candidate lncRNAs by qRT-PCR across early stages of iN cell reprogramming and mouse brain development.
Figure Legend Snippet: QRT-PCR validation of candidate lncRNAs expression. ( A ) Expression detection of candidate lncRNAs by qRT-PCR across early stages of iN cell reprogramming and mouse brain development.

Techniques Used: Quantitative RT-PCR, Expressing

3) Product Images from "Endogenous CHRNA7-ligand SLURP1 as a potential tumor suppressor and anti-nicotinic factor in pancreatic cancer"

Article Title: Endogenous CHRNA7-ligand SLURP1 as a potential tumor suppressor and anti-nicotinic factor in pancreatic cancer

Journal: Oncotarget

doi: 10.18632/oncotarget.24312

High level of circulating SLURP1 is associated with better survival in operable PDAC patients ( A ) The measurement of pancreatic SLURP1 mRNA expression with qRT-PCR in 66 pancreatic samples revealed the lack of expression in non-malignant pancreatic tissues and most PDAC lesions. ( B ) The concentration of SLURP1 was measured in 106 human serum samples using a commercial EIA kit produced by CUSABIO. Multiple comparison testing revealed a lack of difference between the analyzed groups ( p = 0.643). ( C ) Circulating SLURP1 levels correlated with the grade of differentiation but not with any of the TNM parameters. ( D ) Dividing the PDAC patients with resected tumors into SLURP1high/low groups according to the preoperative level of circulating SLURP1 for the Kaplan-Meier survival analysis revealed significantly longer survival of SLURP1high patients (cut-off = 16 ng/ml; log-rank test p = 0.007), MS: median survival. ( E ) The concentration of SLURP1 was re-measured in 67 human serum samples using a commercial EIA kit produced by USCN and confirmed the lack of difference between the analyzed groups ( p = 0.951). ( F ) Nevertheless, the USCN-EIA recognized the CUSABIO standard but not vice versa, and the USCN values for SLURP1 were three-fold lower and lacked any correlation with the CUSABIO values. Additional information is presented in Table . ( G ) Clustering analysis of the RNAseq data obtained for the PDAC specimens corresponding to the serum samples with low and high SLURP1 content.
Figure Legend Snippet: High level of circulating SLURP1 is associated with better survival in operable PDAC patients ( A ) The measurement of pancreatic SLURP1 mRNA expression with qRT-PCR in 66 pancreatic samples revealed the lack of expression in non-malignant pancreatic tissues and most PDAC lesions. ( B ) The concentration of SLURP1 was measured in 106 human serum samples using a commercial EIA kit produced by CUSABIO. Multiple comparison testing revealed a lack of difference between the analyzed groups ( p = 0.643). ( C ) Circulating SLURP1 levels correlated with the grade of differentiation but not with any of the TNM parameters. ( D ) Dividing the PDAC patients with resected tumors into SLURP1high/low groups according to the preoperative level of circulating SLURP1 for the Kaplan-Meier survival analysis revealed significantly longer survival of SLURP1high patients (cut-off = 16 ng/ml; log-rank test p = 0.007), MS: median survival. ( E ) The concentration of SLURP1 was re-measured in 67 human serum samples using a commercial EIA kit produced by USCN and confirmed the lack of difference between the analyzed groups ( p = 0.951). ( F ) Nevertheless, the USCN-EIA recognized the CUSABIO standard but not vice versa, and the USCN values for SLURP1 were three-fold lower and lacked any correlation with the CUSABIO values. Additional information is presented in Table . ( G ) Clustering analysis of the RNAseq data obtained for the PDAC specimens corresponding to the serum samples with low and high SLURP1 content.

Techniques Used: Expressing, Quantitative RT-PCR, Concentration Assay, Enzyme-linked Immunosorbent Assay, Produced, Mass Spectrometry

Preservation of CHRNA7 expression in the pancreas is associated with better prognosis for operable PDAC patients ( A ) CHRNA7 mRNA expression was determined by qRT-PCR in 66 pancreatic samples obtained from the organ donors and patients with chronic pancreatitis (CP) or pancreatic adenocarcinoma (PDAC). The groups were compared using the Kruskal-Wallis test ( p = 0.004) with Dunn’s procedure, which established significant down-regulation of CHRNA7 in PDAC patients ( p < 0.05). ( B ) Pancreatic CHRNA7 mRNA expression is not associated with tumor size, metastasis to lymph nodes, or differentiation grade. ( C ) Dividing the PDAC patients with resected tumors into CHRNAhigh/low groups according to the mRNA level in the Kaplan-Meier survival analysis revealed longer survival in the CHRNAhigh group (cut-off = 50 transcripts/10k PPIB; log-rank test p = 0.048), MS: median survival. ( D ) Specific contribution of the major cell types populating cancerous pancreatic lesion to overall CHRNA7 mRNA expression was evaluated by qRT-PCR analysis of the primary cell cultures (acinar, stellate/stromal, immune) and established cell lines (endothelial HUVEC/HMDEC, pancreatic ductal normal HPDE and tumor AsPC-1, BxPC-3, Capan-1, COLO357, MiaPaca2, PANC-1, SU.86.86, T3M4). ( E ) Immunohistochemical analysis located the CHRNA7 protein in pancreatic islets, immune cells, normal ducts, and cancer cells. Neuronal staining in human brain tissue served as a positive control.
Figure Legend Snippet: Preservation of CHRNA7 expression in the pancreas is associated with better prognosis for operable PDAC patients ( A ) CHRNA7 mRNA expression was determined by qRT-PCR in 66 pancreatic samples obtained from the organ donors and patients with chronic pancreatitis (CP) or pancreatic adenocarcinoma (PDAC). The groups were compared using the Kruskal-Wallis test ( p = 0.004) with Dunn’s procedure, which established significant down-regulation of CHRNA7 in PDAC patients ( p < 0.05). ( B ) Pancreatic CHRNA7 mRNA expression is not associated with tumor size, metastasis to lymph nodes, or differentiation grade. ( C ) Dividing the PDAC patients with resected tumors into CHRNAhigh/low groups according to the mRNA level in the Kaplan-Meier survival analysis revealed longer survival in the CHRNAhigh group (cut-off = 50 transcripts/10k PPIB; log-rank test p = 0.048), MS: median survival. ( D ) Specific contribution of the major cell types populating cancerous pancreatic lesion to overall CHRNA7 mRNA expression was evaluated by qRT-PCR analysis of the primary cell cultures (acinar, stellate/stromal, immune) and established cell lines (endothelial HUVEC/HMDEC, pancreatic ductal normal HPDE and tumor AsPC-1, BxPC-3, Capan-1, COLO357, MiaPaca2, PANC-1, SU.86.86, T3M4). ( E ) Immunohistochemical analysis located the CHRNA7 protein in pancreatic islets, immune cells, normal ducts, and cancer cells. Neuronal staining in human brain tissue served as a positive control.

Techniques Used: Preserving, Expressing, Quantitative RT-PCR, Mass Spectrometry, Immunohistochemistry, Staining, Positive Control

4) Product Images from "Oocyte-specific deletion of furin leads to female infertility by causing early secondary follicle arrest in mice"

Article Title: Oocyte-specific deletion of furin leads to female infertility by causing early secondary follicle arrest in mice

Journal: Cell Death & Disease

doi: 10.1038/cddis.2017.231

Oocyte-specific deletion of furin causes loss of the mature form of ADAMTS1 in oocytes. ( a ) qRT-PCR analysis of the mRNA expression of ADAMTS-1 in oocytes with the indicated genotypes. ** P
Figure Legend Snippet: Oocyte-specific deletion of furin causes loss of the mature form of ADAMTS1 in oocytes. ( a ) qRT-PCR analysis of the mRNA expression of ADAMTS-1 in oocytes with the indicated genotypes. ** P

Techniques Used: Quantitative RT-PCR, Expressing

Localization and oocyte-specific deletion of furin . ( a ) Representative images of subcellular localization of FURIN in GV oocytes. Oocytes were immunolabeled with FURIN antibody (green) and counterstained with propidium iodide (PI) (red) for DNA. GV, germinal vesicle. Scale bar=20 μ m. ( b ) FURIN IHC staining showing the localization during follicular development in the mouse ovary. Scale bar=100 μ m. ( c ) qRT-PCR showing furin mRNA level in fur fl/fl and fur fl/fl ; Zp3-Cre oocytes, respectively ( n =3 for each genotype). ** P
Figure Legend Snippet: Localization and oocyte-specific deletion of furin . ( a ) Representative images of subcellular localization of FURIN in GV oocytes. Oocytes were immunolabeled with FURIN antibody (green) and counterstained with propidium iodide (PI) (red) for DNA. GV, germinal vesicle. Scale bar=20 μ m. ( b ) FURIN IHC staining showing the localization during follicular development in the mouse ovary. Scale bar=100 μ m. ( c ) qRT-PCR showing furin mRNA level in fur fl/fl and fur fl/fl ; Zp3-Cre oocytes, respectively ( n =3 for each genotype). ** P

Techniques Used: Immunolabeling, Immunohistochemistry, Staining, Quantitative RT-PCR

5) Product Images from "Genome-wide identification and characterization of miRNAome from tomato (Solanum lycopersicum) roots and root-knot nematode (Meloidogyne incognita) during susceptible interaction"

Article Title: Genome-wide identification and characterization of miRNAome from tomato (Solanum lycopersicum) roots and root-knot nematode (Meloidogyne incognita) during susceptible interaction

Journal: PLoS ONE

doi: 10.1371/journal.pone.0175178

Relative expression analysis of selected conserved and novel tomato miRNAs and their targets through qRT-PCR at four stages of disease development during tomato-RKN susceptible interaction. The correlation in expression profile was deciphered between conserved miRNAs including, miR156(i), miR164(i), miR159(i), miR168(i) and miR396(i) and their target genes, SBP , NAC , GAMYB-like ( MYB33 and MYB65 ), AGO1 and GRF1 , respectively. The correlation in expression profile of novel miRNA, Sly_miRNA996 with its target, MYB-like transcription factor gene was also determined. For qRT-PCR analysis, two technical replicates for each of three biological replicates were used. Fold change was calculated through delta delta Ct method that represents the change in expression level of miRNA and target gene in the infected sample relative to the uninfected control. The fold change of uninfected sample of each stage was taken as 1 and presented with solid line. The data is presented as the mean of three biological replicates ±standard error of the mean. S1-Stage 1, S2-Stage 2, S3-Stage 3, S5-Stage 5, UI-Uninfected sample, I-Infected sample.
Figure Legend Snippet: Relative expression analysis of selected conserved and novel tomato miRNAs and their targets through qRT-PCR at four stages of disease development during tomato-RKN susceptible interaction. The correlation in expression profile was deciphered between conserved miRNAs including, miR156(i), miR164(i), miR159(i), miR168(i) and miR396(i) and their target genes, SBP , NAC , GAMYB-like ( MYB33 and MYB65 ), AGO1 and GRF1 , respectively. The correlation in expression profile of novel miRNA, Sly_miRNA996 with its target, MYB-like transcription factor gene was also determined. For qRT-PCR analysis, two technical replicates for each of three biological replicates were used. Fold change was calculated through delta delta Ct method that represents the change in expression level of miRNA and target gene in the infected sample relative to the uninfected control. The fold change of uninfected sample of each stage was taken as 1 and presented with solid line. The data is presented as the mean of three biological replicates ±standard error of the mean. S1-Stage 1, S2-Stage 2, S3-Stage 3, S5-Stage 5, UI-Uninfected sample, I-Infected sample.

Techniques Used: Expressing, Quantitative RT-PCR, Infection

Expression analysis of tomato miRNAs through qRT-PCR at four stages of disease development during susceptible response (A-C) and at two stages of disease development during resistance response (D). To normalize the expression level, 18S rRNA was selected as internal control. All the experiments were conducted using two technical replicates for each of three biological replicates. Fold change was calculated through delta delta Ct method that represents the change in expression level in the infected sample relative to uninfected control sample. Data is the average of three biological replicates ± standard error of the mean. The student’s t-test (P
Figure Legend Snippet: Expression analysis of tomato miRNAs through qRT-PCR at four stages of disease development during susceptible response (A-C) and at two stages of disease development during resistance response (D). To normalize the expression level, 18S rRNA was selected as internal control. All the experiments were conducted using two technical replicates for each of three biological replicates. Fold change was calculated through delta delta Ct method that represents the change in expression level in the infected sample relative to uninfected control sample. Data is the average of three biological replicates ± standard error of the mean. The student’s t-test (P

Techniques Used: Expressing, Quantitative RT-PCR, Infection

6) Product Images from "Quantitative Real-Time PCR for Determination of Microcystin Synthetase E Copy Numbers for Microcystis and Anabaena in Lakes"

Article Title: Quantitative Real-Time PCR for Determination of Microcystin Synthetase E Copy Numbers for Microcystis and Anabaena in Lakes

Journal:

doi: 10.1128/AEM.69.12.7289-7297.2003

Microcystin concentration and Anabaena and Microcystis microcystin synthetase E ( mcyE ) copy numbers in samples of lake water collected from different depths of four Lake Hiidenvesi basins on 15 August 2001. Microcystin concentration (×) (in micrograms per liter) was determined by an ELISA. Microcystis mcyE copy numbers (number of copies per milliliter) were obtained by QRT PCR and were calculated with the external standards of Microcystis sp. strain PCC 7941 (•), Microcystis sp. strain PCC 7806 (○), Anabaena sp. strain 202A1 (▪), and Anabaena sp. strain 315 (□).
Figure Legend Snippet: Microcystin concentration and Anabaena and Microcystis microcystin synthetase E ( mcyE ) copy numbers in samples of lake water collected from different depths of four Lake Hiidenvesi basins on 15 August 2001. Microcystin concentration (×) (in micrograms per liter) was determined by an ELISA. Microcystis mcyE copy numbers (number of copies per milliliter) were obtained by QRT PCR and were calculated with the external standards of Microcystis sp. strain PCC 7941 (•), Microcystis sp. strain PCC 7806 (○), Anabaena sp. strain 202A1 (▪), and Anabaena sp. strain 315 (□).

Techniques Used: Concentration Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Periodic Counter-current Chromatography

Microcystin concentration and Anabaena and Microcystis microcystin synthetase gene E ( mcyE ) copy numbers using Lake Tuusulanjärvi water samples collected during the summer of 1999. Microcystin concentration (×) (in micrograms per liter) was determined by an ELISA. Microcystis mcyE copy numbers (number of copies per milliliter) were obtained by QRT-PCR and were calculated with the external standards of Microcystis sp. strain PCC 7941 (•), Microcystis sp. strain PCC 7806 (○), Anabaena sp. strain 202A1 (▪), and Anabaena sp. strain 315 (□).
Figure Legend Snippet: Microcystin concentration and Anabaena and Microcystis microcystin synthetase gene E ( mcyE ) copy numbers using Lake Tuusulanjärvi water samples collected during the summer of 1999. Microcystin concentration (×) (in micrograms per liter) was determined by an ELISA. Microcystis mcyE copy numbers (number of copies per milliliter) were obtained by QRT-PCR and were calculated with the external standards of Microcystis sp. strain PCC 7941 (•), Microcystis sp. strain PCC 7806 (○), Anabaena sp. strain 202A1 (▪), and Anabaena sp. strain 315 (□).

Techniques Used: Concentration Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Periodic Counter-current Chromatography

Ct values obtained by microcystin synthetase gene E ( mcyE ) QRT-PCR with Microcystis and Anabaena strains as a function of mcyE copy number. (A) Microcystis sp. strains GL 260735, PCC 7806, and PCC 7941. (B) Anabaena sp. strains 90, 315, and 202A1. Amplification efficiencies, e ( e = 10−1/S − 1, where S is the slope of the linear regression), of the Microcystis and Anabaena mcyE QRT-PCR were also calculated as a function of mcyE copy numbers. Error bars, which are hidden by the symbols in almost all cases, give the standard deviations for three independent amplifications.
Figure Legend Snippet: Ct values obtained by microcystin synthetase gene E ( mcyE ) QRT-PCR with Microcystis and Anabaena strains as a function of mcyE copy number. (A) Microcystis sp. strains GL 260735, PCC 7806, and PCC 7941. (B) Anabaena sp. strains 90, 315, and 202A1. Amplification efficiencies, e ( e = 10−1/S − 1, where S is the slope of the linear regression), of the Microcystis and Anabaena mcyE QRT-PCR were also calculated as a function of mcyE copy numbers. Error bars, which are hidden by the symbols in almost all cases, give the standard deviations for three independent amplifications.

Techniques Used: Quantitative RT-PCR, Periodic Counter-current Chromatography, Amplification

7) Product Images from "Production of Infectious Genotype 1b Virus Particles in Cell Culture and Impairment by Replication Enhancing Mutations"

Article Title: Production of Infectious Genotype 1b Virus Particles in Cell Culture and Impairment by Replication Enhancing Mutations

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1000475

In vivo infectivity of Con1/wt, Con1/K1846T and Con1/NS3+K1846T genomes in uPA-SCID mice. Huh7-Lunet cells were transfected with either of these constructs, supernatants were collected 12 and 24 h post transfection, pooled for each construct and used for virus purification and concentration as described in Materials and Methods . Two mice were each inoculated with 2×10 8 IU HCV RNA per mouse and construct (100 µl inoculum size) and viral RNA loads in sera were determined at the indicated time points after inoculation by qRT-PCR. In case of Con1/K1846T inoculated mice, one died at week 2 (not shown) and the second shortly after week 6. While sera of Con1/wt and Con1/K1846T inoculated mice contained high viral loads already in the first blood sample, Con1/NS3+K1846T-inoculated mice remained HCV RNA negative throughout the 10 weeks observation period.
Figure Legend Snippet: In vivo infectivity of Con1/wt, Con1/K1846T and Con1/NS3+K1846T genomes in uPA-SCID mice. Huh7-Lunet cells were transfected with either of these constructs, supernatants were collected 12 and 24 h post transfection, pooled for each construct and used for virus purification and concentration as described in Materials and Methods . Two mice were each inoculated with 2×10 8 IU HCV RNA per mouse and construct (100 µl inoculum size) and viral RNA loads in sera were determined at the indicated time points after inoculation by qRT-PCR. In case of Con1/K1846T inoculated mice, one died at week 2 (not shown) and the second shortly after week 6. While sera of Con1/wt and Con1/K1846T inoculated mice contained high viral loads already in the first blood sample, Con1/NS3+K1846T-inoculated mice remained HCV RNA negative throughout the 10 weeks observation period.

Techniques Used: In Vivo, Infection, Mouse Assay, Transfection, Construct, Purification, Concentration Assay, Quantitative RT-PCR

Capture of Con1 particles by E2-specific antibodies and characterization of captured particles. (A) Identification of monoclonal antibodies reacting with Con1 envelope glycoproteins. HCVpp carrying Con1 envelope glycoproteins were incubated with immobilized antibodies specified in the bottom and captured particles were quantitated by using p24-specific ELISA. Numbers above each bar refer to fold increase above background as determined by capture assay with an irrelavant antibody (RO4). Mean values of three experiments and the standard deviations are given. (B) Capture of HCV particles from supernatants of Huh-7 cells transfected with Con1/wt or Con1/K1846T or the Con1 mutant lacking most of the envelope glycoprotein coding region (ΔE1–E2). Bound particles were quantified by using TaqMan qRT-PCR. Mean values of triplicate measurements including the standard error of the means are given. (C) Characterization of captured Con1/wt particles by nuclease treatment. Concentrated culture supernatant of Con1/wt transfected cells was used for capture with control antibody R04 (right bar) or CBH-5 (all other bars). Immune complexes obtained with the latter were split into 4 aliquots that were left untreated or treated with 0.5% Triton X-100, or nuclease S7, or 0.5% Triton X-100 and S7 nuclease. RNA was extracted and quantified by TaqMan qRT-PCR.
Figure Legend Snippet: Capture of Con1 particles by E2-specific antibodies and characterization of captured particles. (A) Identification of monoclonal antibodies reacting with Con1 envelope glycoproteins. HCVpp carrying Con1 envelope glycoproteins were incubated with immobilized antibodies specified in the bottom and captured particles were quantitated by using p24-specific ELISA. Numbers above each bar refer to fold increase above background as determined by capture assay with an irrelavant antibody (RO4). Mean values of three experiments and the standard deviations are given. (B) Capture of HCV particles from supernatants of Huh-7 cells transfected with Con1/wt or Con1/K1846T or the Con1 mutant lacking most of the envelope glycoprotein coding region (ΔE1–E2). Bound particles were quantified by using TaqMan qRT-PCR. Mean values of triplicate measurements including the standard error of the means are given. (C) Characterization of captured Con1/wt particles by nuclease treatment. Concentrated culture supernatant of Con1/wt transfected cells was used for capture with control antibody R04 (right bar) or CBH-5 (all other bars). Immune complexes obtained with the latter were split into 4 aliquots that were left untreated or treated with 0.5% Triton X-100, or nuclease S7, or 0.5% Triton X-100 and S7 nuclease. RNA was extracted and quantified by TaqMan qRT-PCR.

Techniques Used: Incubation, Enzyme-linked Immunosorbent Assay, Transfection, Mutagenesis, Quantitative RT-PCR

8) Product Images from "Time-Dependent Effects of POT1 Knockdown on Proliferation, Tumorigenicity, and HDACi Response of SK-OV3 Ovarian Cancer Cells"

Article Title: Time-Dependent Effects of POT1 Knockdown on Proliferation, Tumorigenicity, and HDACi Response of SK-OV3 Ovarian Cancer Cells

Journal: BioMed Research International

doi: 10.1155/2018/7184253

POT1 mRNA expression as determined by qRT-PCR (a) and POT1 protein expression as determined by western blot analysis (b) in shRNA-infected human ovarian cancer SK-OV3 cells. POT1-KD significantly decreased cell proliferation (c) and colony formation (d) in the early POT1-KD SK-OV3 cells. “Negative control” indicates negative control SK-OV3 cells that express a nonspecific shRNA, and “POT1-KD” represents POT1-knockdown SK-OV3 cells that express POT1-specific shRNA. (c) shows the cPDs of the SK-OV3 cells. The cPDs of the negative control cells are depicted with black dots and lines, while the cPDs of the immediate effect POT1-KD cells are depicted with black squares and lines. (d) shows images from the soft agar colony formation assay that used SK-OV3 cells. The dark dots represent the colonies. Images of the negative control cells and the immediate effect POT1-KD cells were captured via digital phase-contrast microscopy at 4x magnification; scale bar: 1000 μ m. ∗∗ P
Figure Legend Snippet: POT1 mRNA expression as determined by qRT-PCR (a) and POT1 protein expression as determined by western blot analysis (b) in shRNA-infected human ovarian cancer SK-OV3 cells. POT1-KD significantly decreased cell proliferation (c) and colony formation (d) in the early POT1-KD SK-OV3 cells. “Negative control” indicates negative control SK-OV3 cells that express a nonspecific shRNA, and “POT1-KD” represents POT1-knockdown SK-OV3 cells that express POT1-specific shRNA. (c) shows the cPDs of the SK-OV3 cells. The cPDs of the negative control cells are depicted with black dots and lines, while the cPDs of the immediate effect POT1-KD cells are depicted with black squares and lines. (d) shows images from the soft agar colony formation assay that used SK-OV3 cells. The dark dots represent the colonies. Images of the negative control cells and the immediate effect POT1-KD cells were captured via digital phase-contrast microscopy at 4x magnification; scale bar: 1000 μ m. ∗∗ P

Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, shRNA, Infection, Negative Control, Soft Agar Assay, Microscopy

9) Product Images from "Time-Dependent Effects of POT1 Knockdown on Proliferation, Tumorigenicity, and HDACi Response of SK-OV3 Ovarian Cancer Cells"

Article Title: Time-Dependent Effects of POT1 Knockdown on Proliferation, Tumorigenicity, and HDACi Response of SK-OV3 Ovarian Cancer Cells

Journal: BioMed Research International

doi: 10.1155/2018/7184253

POT1 mRNA expression as determined by qRT-PCR (a) and POT1 protein expression as determined by western blot analysis (b) in shRNA-infected human ovarian cancer SK-OV3 cells. POT1-KD significantly decreased cell proliferation (c) and colony formation (d) in the early POT1-KD SK-OV3 cells. “Negative control” indicates negative control SK-OV3 cells that express a nonspecific shRNA, and “POT1-KD” represents POT1-knockdown SK-OV3 cells that express POT1-specific shRNA. (c) shows the cPDs of the SK-OV3 cells. The cPDs of the negative control cells are depicted with black dots and lines, while the cPDs of the immediate effect POT1-KD cells are depicted with black squares and lines. (d) shows images from the soft agar colony formation assay that used SK-OV3 cells. The dark dots represent the colonies. Images of the negative control cells and the immediate effect POT1-KD cells were captured via digital phase-contrast microscopy at 4x magnification; scale bar: 1000 μ m. ∗∗ P < 0.01, compared with negative control cells.
Figure Legend Snippet: POT1 mRNA expression as determined by qRT-PCR (a) and POT1 protein expression as determined by western blot analysis (b) in shRNA-infected human ovarian cancer SK-OV3 cells. POT1-KD significantly decreased cell proliferation (c) and colony formation (d) in the early POT1-KD SK-OV3 cells. “Negative control” indicates negative control SK-OV3 cells that express a nonspecific shRNA, and “POT1-KD” represents POT1-knockdown SK-OV3 cells that express POT1-specific shRNA. (c) shows the cPDs of the SK-OV3 cells. The cPDs of the negative control cells are depicted with black dots and lines, while the cPDs of the immediate effect POT1-KD cells are depicted with black squares and lines. (d) shows images from the soft agar colony formation assay that used SK-OV3 cells. The dark dots represent the colonies. Images of the negative control cells and the immediate effect POT1-KD cells were captured via digital phase-contrast microscopy at 4x magnification; scale bar: 1000 μ m. ∗∗ P < 0.01, compared with negative control cells.

Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, shRNA, Infection, Negative Control, Soft Agar Assay, Microscopy

10) Product Images from "Biomarker for Spinal Muscular Atrophy: Expression of SMN in Peripheral Blood of SMA Patients and Healthy Controls"

Article Title: Biomarker for Spinal Muscular Atrophy: Expression of SMN in Peripheral Blood of SMA Patients and Healthy Controls

Journal: PLoS ONE

doi: 10.1371/journal.pone.0139950

Expression of SMN2 mRNA in SMA patients and healthy controls. SMN2 mRNA was isolated from blood and analyzed using qRT-PCR. Expression level is calculated using 2ˆ-deltaCp of the reference gene. There is a strong overlap of SMN2 mRNA in the different patient groups. Note that in the healthy controls SMN2 levels are lower than levels in patients with the same SMN2 copy number.
Figure Legend Snippet: Expression of SMN2 mRNA in SMA patients and healthy controls. SMN2 mRNA was isolated from blood and analyzed using qRT-PCR. Expression level is calculated using 2ˆ-deltaCp of the reference gene. There is a strong overlap of SMN2 mRNA in the different patient groups. Note that in the healthy controls SMN2 levels are lower than levels in patients with the same SMN2 copy number.

Techniques Used: Expressing, Isolation, Quantitative RT-PCR

11) Product Images from "Carnosine Prevents Aβ-Induced Oxidative Stress and Inflammation in Microglial Cells: A Key Role of TGF-β1"

Article Title: Carnosine Prevents Aβ-Induced Oxidative Stress and Inflammation in Microglial Cells: A Key Role of TGF-β1

Journal: Cells

doi: 10.3390/cells8010064

Carnosine suppresses the Aβ1-42-induced mRNA expression levels of iNOS, Nox1, and Nox2 and increases the expression of TGF-β1 mRNA. Effects of Aβ1-42 and carnosine (Aβ1-42 + Car (co-treat.) and Aβ1-42 + Car (co-inc.)) on ( A ) iNOS, ( B ) Nox1, ( C ) Nox2, and ( D ) TGF-β1 mRNAs expression were examined by qRT-PCR. The abundance of each mRNA of interest was expressed relative to the abundance of GAPDH-mRNA, as an internal control. As a negative control, a reaction in the absence of cDNA (no template control, NTC) was performed. qRT-PCR amplifications were performed in quadruplicate. Standard deviations are represented by vertical bars. * significantly different from resting cells, p
Figure Legend Snippet: Carnosine suppresses the Aβ1-42-induced mRNA expression levels of iNOS, Nox1, and Nox2 and increases the expression of TGF-β1 mRNA. Effects of Aβ1-42 and carnosine (Aβ1-42 + Car (co-treat.) and Aβ1-42 + Car (co-inc.)) on ( A ) iNOS, ( B ) Nox1, ( C ) Nox2, and ( D ) TGF-β1 mRNAs expression were examined by qRT-PCR. The abundance of each mRNA of interest was expressed relative to the abundance of GAPDH-mRNA, as an internal control. As a negative control, a reaction in the absence of cDNA (no template control, NTC) was performed. qRT-PCR amplifications were performed in quadruplicate. Standard deviations are represented by vertical bars. * significantly different from resting cells, p

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

12) Product Images from "NLRP6, decreased in gastric cancer, suppresses tumorigenicity of gastric cancer cells"

Article Title: NLRP6, decreased in gastric cancer, suppresses tumorigenicity of gastric cancer cells

Journal: Cancer Management and Research

doi: 10.2147/CMAR.S182980

Effects of NLRP6 on STAT3 signaling. Notes: ( A ) Immunoblot of phosphorylated STAT3, STAT3, Bcl-2, and MMP-2. Blots are representative of three separate experiments. ( B ) mRNA levels of Bcl-2 and MMP-2 were assessed by qRT-PCR. ( C ) BGC-823 and HGC-27 cells were transfected with a Bcl-2 or an MMP-2 luciferase reporter plasmid. The cells were then cultured for 48 hours before determination of normalized luciferase activity. WT, wild-type cells; vector, cells stably expressed control vector; NLR6, cells stably expressed NLRP6. *** P
Figure Legend Snippet: Effects of NLRP6 on STAT3 signaling. Notes: ( A ) Immunoblot of phosphorylated STAT3, STAT3, Bcl-2, and MMP-2. Blots are representative of three separate experiments. ( B ) mRNA levels of Bcl-2 and MMP-2 were assessed by qRT-PCR. ( C ) BGC-823 and HGC-27 cells were transfected with a Bcl-2 or an MMP-2 luciferase reporter plasmid. The cells were then cultured for 48 hours before determination of normalized luciferase activity. WT, wild-type cells; vector, cells stably expressed control vector; NLR6, cells stably expressed NLRP6. *** P

Techniques Used: Quantitative RT-PCR, Transfection, Luciferase, Plasmid Preparation, Cell Culture, Activity Assay, Stable Transfection

13) Product Images from "Constitutive Expression of Yes-Associated Protein (Yap) in Adult Skeletal Muscle Fibres Induces Muscle Atrophy and Myopathy"

Article Title: Constitutive Expression of Yes-Associated Protein (Yap) in Adult Skeletal Muscle Fibres Induces Muscle Atrophy and Myopathy

Journal: PLoS ONE

doi: 10.1371/journal.pone.0059622

Up regulation in genes associated with muscle regeneration and atrophy in MCK-tTA-hYAP1 S127A mice. TA muscles of transgenic mice were harvested following doxycycline withdrawal at indicated time points and RNA processed for qRT-PCR analysis of A) hYAP1 mRNA, B) embryonic myosin heavy chain, C) Myf5 mRNA, D) Pax7 mRNA, E) myogenin mRNA, F) Caspase-3 mRNA, G) Atrogin-1 mRNA and H) MuRF-1 mRNA. Expression normalised to Gapdh. All values present mean ±SEM (n = 12) and are displayed as fold change relative to control. ***P
Figure Legend Snippet: Up regulation in genes associated with muscle regeneration and atrophy in MCK-tTA-hYAP1 S127A mice. TA muscles of transgenic mice were harvested following doxycycline withdrawal at indicated time points and RNA processed for qRT-PCR analysis of A) hYAP1 mRNA, B) embryonic myosin heavy chain, C) Myf5 mRNA, D) Pax7 mRNA, E) myogenin mRNA, F) Caspase-3 mRNA, G) Atrogin-1 mRNA and H) MuRF-1 mRNA. Expression normalised to Gapdh. All values present mean ±SEM (n = 12) and are displayed as fold change relative to control. ***P

Techniques Used: Mouse Assay, Transgenic Assay, Quantitative RT-PCR, Expressing

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Amplification:

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Positive Control:

Article Title: Endogenous CHRNA7-ligand SLURP1 as a potential tumor suppressor and anti-nicotinic factor in pancreatic cancer
Article Snippet: Esophageal RNA was purchased from BioCat (Heidelberg, Germany) and was used as a positive control for SLURP1 expression. .. All instruments and reagents for qRT-PCR analysis were purchased from Roche Applied Biosciences AG (Mannheim, Germany) and applied as described previously [ , ].

Synthesized:

Article Title: Time-Dependent Effects of POT1 Knockdown on Proliferation, Tumorigenicity, and HDACi Response of SK-OV3 Ovarian Cancer Cells
Article Snippet: The cells were harvested, and total RNA was extracted using an RNeasy Plus Mini Kit (Qiagen, Germantown, MD, USA) according to the manufacturer's instructions. cDNA was synthesized with 3.0 μ g of RNA and a Superscript III kit (Invitrogen, USA) according to the manufacturer's protocol and was stored at −20°C until use. qRT-PCR was performed in a Bio-Rad CFX96 Real-Time System (Bio-Rad, Singapore, USA). .. The primers indicated below, which were specific for hTERT mRNA and the TaqMan probe [ ], were also used in the qRT-PCR assays (Roche, Indianapolis, IN, USA): forward 5′-TGACACCTCACCTCACCCAC-3′ and reverse 5′-CACTGTCTTCCGCAAGTTCAC-3′ and TaqMan probe 5′-ACCCTGGTCCGAGGTGTCCCTGAG-3′.

Quantitative RT-PCR:

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Article Title: Genome-wide identification and characterization of miRNAome from tomato (Solanum lycopersicum) roots and root-knot nematode (Meloidogyne incognita) during susceptible interaction
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Article Title: Production of Infectious Genotype 1b Virus Particles in Cell Culture and Impairment by Replication Enhancing Mutations
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Article Title: Time-Dependent Effects of POT1 Knockdown on Proliferation, Tumorigenicity, and HDACi Response of SK-OV3 Ovarian Cancer Cells
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Article Title: Constitutive Expression of Yes-Associated Protein (Yap) in Adult Skeletal Muscle Fibres Induces Muscle Atrophy and Myopathy
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Article Title: Involvement of the Interleukin-23/Interleukin-17 Axis in Chronic Hepatitis C Virus Infection and Its Treatment Responses
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Article Title: αII-spectrin and βII-spectrin do not affect TGFβ1-induced myofibroblast differentiation
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SYBR Green Assay:

Article Title: Quantitative Real-Time PCR for Determination of Microcystin Synthetase E Copy Numbers for Microcystis and Anabaena in Lakes
Article Snippet: The QRT-PCR was performed with 1 μl of DNA from a standard strain or lake water sample, 3 mM MgCl2 , 0.5 μM concentrations of both primers (Sigma-Genosys, Ltd.), and 1 μl of hot start reaction mix to a final volume of 10 μl (LightCycler fastStart DNA master SYBR green I kit; Roche Diagnostics). .. Generation of the products was monitored after each extension step at 78°C in Microcystis and 77°C in Anabaena mcyE QRT-PCR by measuring the fluorescence of double-stranded DNA binding SYBR green 1 dye using LightCycler QRT-PCR (Roche Diagnostics). .. All lake water samples were amplified in triplicate.

Article Title: Circulating microRNAs -192 and -194 are associated with the presence and incidence of diabetes mellitus
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Article Title: The Utilization of RNA Silencing Technology to Mitigate the Voriconazole Resistance of Aspergillus Flavus; Lipofectamine-Based Delivery
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Article Title: αII-spectrin and βII-spectrin do not affect TGFβ1-induced myofibroblast differentiation
Article Snippet: To assess gene expression, the RNA was reverse transcribed using the First Strand cDNA synthesis kit (Thermo Fisher Scientific) using random hexamer primers in accordance with the manufacturer’s instructions. .. Gene expression quantification was performed using qRT-PCR analysis and SYBR Green Supermix (Roche, Basel, Switzerland). .. The thermal cycling conditions were 2 min at 95 °C (enzyme activation), followed by 15 s at 95 °C, 30 s at 60 °C, and 30 s at 72 °C (40 cycles).

Article Title: Interleukin-33 (IL-33) Increases Hyperoxia-Induced Bronchopulmonary Dysplasia in Newborn Mice by Regulation of Inflammatory Mediators
Article Snippet: Total RNA was extracted from cells of the lungs from the three mouse groups using Trizol reagent. .. The levels of IL-33, BCL-2, BAX, IL-1β, CXCL-1, MCP-1, TGF-β, and SMAD-7 mRNA were determined using SYBR Green and a LightCycler 480 quantitative real-time polymerase chain reaction (qRT-PCR) system (Roche). .. GAPDH was used as a control.

Random Hexamer Labeling:

Article Title: αII-spectrin and βII-spectrin do not affect TGFβ1-induced myofibroblast differentiation
Article Snippet: To assess gene expression, the RNA was reverse transcribed using the First Strand cDNA synthesis kit (Thermo Fisher Scientific) using random hexamer primers in accordance with the manufacturer’s instructions. .. Gene expression quantification was performed using qRT-PCR analysis and SYBR Green Supermix (Roche, Basel, Switzerland).

Expressing:

Article Title: Endogenous CHRNA7-ligand SLURP1 as a potential tumor suppressor and anti-nicotinic factor in pancreatic cancer
Article Snippet: Esophageal RNA was purchased from BioCat (Heidelberg, Germany) and was used as a positive control for SLURP1 expression. .. All instruments and reagents for qRT-PCR analysis were purchased from Roche Applied Biosciences AG (Mannheim, Germany) and applied as described previously [ , ].

Article Title: Genome-wide identification and characterization of miRNAome from tomato (Solanum lycopersicum) roots and root-knot nematode (Meloidogyne incognita) during susceptible interaction
Article Snippet: The polyadenylated product was purified and reverse transcribed using a poly-T adapter and SuperScript Reverse Transcriptase III (Invitrogen). .. Further, expression of miRNAs was measured through Taqman probe based qRT- PCR method (Roche, Mannheim, Germany). .. The qRT-PCR for expression analysis of miRNAs was performed using miRNA specific forward primer (0.5 μM), universal reverse primer (0.5 μM), TaqMan probe complementary to the poly-T adapter (0.2 μM) with LightCycler® 480 Probe Master Mix.

Article Title: Carnosine Prevents Aβ-Induced Oxidative Stress and Inflammation in Microglial Cells: A Key Role of TGF-β1
Article Snippet: Paragraph title: 2.7. Gene Expression Analysis by Quantitative Real-Time PCR (qRT-PCR) ... Next, each sample was quantified, diluted to a final concentration of 25 ng/µL, and used for qRT-PCR analysis (LightCycler® 480 System, Roche Molecular Systems, Inc., Pleasanton, CA, USA).

Article Title: Circulating microRNAs -192 and -194 are associated with the presence and incidence of diabetes mellitus
Article Snippet: Samples were screened for the expression of 372 miRNAs by qRT-PCR based arrays (Human panel I V2.M, miRCURY LNA SYBR Green Master Mix; Exiqon) on a 7900HT Fast qRT-PCR System (Applied Biosystems, Foster City, California, USA). .. PCR assays were performed in triplicates on a LightCycler 480 II qRT-PCR System (Roche, Rotkreuz, Switzerland) and all miRNAs were detected at Cq < 37.

Article Title: αII-spectrin and βII-spectrin do not affect TGFβ1-induced myofibroblast differentiation
Article Snippet: To assess gene expression, the RNA was reverse transcribed using the First Strand cDNA synthesis kit (Thermo Fisher Scientific) using random hexamer primers in accordance with the manufacturer’s instructions. .. Gene expression quantification was performed using qRT-PCR analysis and SYBR Green Supermix (Roche, Basel, Switzerland). .. The thermal cycling conditions were 2 min at 95 °C (enzyme activation), followed by 15 s at 95 °C, 30 s at 60 °C, and 30 s at 72 °C (40 cycles).

Western Blot:

Article Title: Constitutive Expression of Yes-Associated Protein (Yap) in Adult Skeletal Muscle Fibres Induces Muscle Atrophy and Myopathy
Article Snippet: PCR primers for genotyping, end-point PCR primers for hYAP and Gapdh mRNA and qRT-PCR primers and probes (Roche Universal Probe Library). (DOCX) Click here for additional data file. .. PCR primers for genotyping, end-point PCR primers for hYAP and Gapdh mRNA and qRT-PCR primers and probes (Roche Universal Probe Library). (DOCX) Click here for additional data file.

TUNEL Assay:

Article Title: NLRP6, decreased in gastric cancer, suppresses tumorigenicity of gastric cancer cells
Article Snippet: At 27 days after cell inoculation, the mice were sacrificed and the tumors were collected. .. Tumor xenografts were subjected to qRT-PCR analysis, TUNEL assay (Hoffman-La Roche Ltd), and IHC staining with anti-Ki-67 (Abcam). .. To evaluate the cell migration and invasive capacity of cells, Transwell assays were performed using Matrigel noncoated and Matrigel-coated Boyden chamber (BD Biosciences), respectively.

Flow Cytometry:

Article Title: Biomarker for Spinal Muscular Atrophy: Expression of SMN in Peripheral Blood of SMA Patients and Healthy Controls
Article Snippet: Paragraph title: Supporting Information Transparent reporting of clinical trials CONSORT 2010 Flow Diagram. Comparison of newly developed SMN mRNA and SMN protein assays with previously published assays. Study protocol. Raw data of the assay. TREND Checklist. ... Comparison of Roche qRT-PCR assay on COBAS (SMN2 mRNA assay B) with SMN2 mRNA assay B ([ ]et al).

Sequencing:

Article Title: The Utilization of RNA Silencing Technology to Mitigate the Voriconazole Resistance of Aspergillus Flavus; Lipofectamine-Based Delivery
Article Snippet: Nucleotide sequences of cyp51A (NCBI accession numbers: XM_002375082) and MDR1 (NCBI accession numbers: XM_002382940) were obtained from the published gene sequence of A. flavus NRRL3357 (http://www.ncbi.nlm.nih.gov/pubmed/). .. Cyp51A and MDR1 mRNA levels were amplified by qRT-PCR instrument (light cycler 96, Roche) using SYBR Green Master Mix (AMPLIQON, Denmark).

Concentration Assay:

Article Title: Quantitative Real-Time PCR for Determination of Microcystin Synthetase E Copy Numbers for Microcystis and Anabaena in Lakes
Article Snippet: The mcyE copy numbers of the DNAs of the standard strains were calculated using the following equation assuming that each genome had only one mcyE gene and that the molecular weight of 1 bp was 660 g mol−1 : number of copies per microliter = (6 × 1023 )(DNA concentration)/molecular weight of one genome, where 6 × 1023 is the number of copies per mole, the DNA concentration is given in grams per microliter, and the molecular weight of one genome is given in grams per mole. .. Generation of the products was monitored after each extension step at 78°C in Microcystis and 77°C in Anabaena mcyE QRT-PCR by measuring the fluorescence of double-stranded DNA binding SYBR green 1 dye using LightCycler QRT-PCR (Roche Diagnostics).

Article Title: Carnosine Prevents Aβ-Induced Oxidative Stress and Inflammation in Microglial Cells: A Key Role of TGF-β1
Article Snippet: Reverse transcription was performed using 100 ng of total RNA, RNaseH reverse transcriptase, and random primer hexamers (Superscript II, Thermo Fisher Scientific). .. Next, each sample was quantified, diluted to a final concentration of 25 ng/µL, and used for qRT-PCR analysis (LightCycler® 480 System, Roche Molecular Systems, Inc., Pleasanton, CA, USA). .. The QuantiTect Primer Assays (Qiagen) employed for the gene expression analysis along with the official name, official symbol, alternative titles/symbols, detected transcript, amplicon length, and primers catalog number are shown in . qRT-PCR amplifications were performed in quadruplicate using a mixture of SYBR Green PCR Master Mix (Thermo Fisher Scientific), cDNA samples (100 ng), and specific primers (total reaction volume of 10 μL).

Article Title: αII-spectrin and βII-spectrin do not affect TGFβ1-induced myofibroblast differentiation
Article Snippet: RNA concentration and purity were determined by UV spectrophotometry (NanoDrop Technologies, Wilmington, NC). .. Gene expression quantification was performed using qRT-PCR analysis and SYBR Green Supermix (Roche, Basel, Switzerland).

Infection:

Article Title: Production of Infectious Genotype 1b Virus Particles in Cell Culture and Impairment by Replication Enhancing Mutations
Article Snippet: Inocula contained 2×108 IU HCV RNA for each preparation and 540 pg core protein for Con1/wt, 200 pg for Con1/K1846T and 23 pg core protein for Con1/NS3+K1846T. .. EDTA plasma samples were collected at weekly intervals after inoculation and infection was monitored by a commercial qRT-PCR kit (Roche COBAS AmpliPrep/TaqMan48 assay, Roche Diagnostics). .. Due to dilution of the samples, the detection limit of the test was 750 IU/ml.

Isolation:

Article Title: The novel lncRNA lnc-NR2F1 is pro-neurogenic and mutated in human neurodevelopmental disorders
Article Snippet: Isolated RNA was subjected to DNAse treatment using TurboDNase and purified by phenol-chloroform extraction and ethanol precipitation. .. For qRT-PCR analysis we used Roche’s Lightcycler and Stratagene’s RT kit.

Article Title: Oocyte-specific deletion of furin leads to female infertility by causing early secondary follicle arrest in mice
Article Snippet: In each section, only those follicles in which the nucleus of the oocyte was clearly visible were scored and the cumulative follicle counts were multiplied by a correction factor of 5 to represent the estimated number of total follicles in an ovary. .. RNA was isolated from ~100 oocytes for each group and reverse transcription reaction was carried out using the RNeasy Kit (Qiagen, Hilden, Germany). mRNA level of each gene was validated by qRT-PCR analysis (Roche; 480, Boehringer, Mannheim, Germany) according to the manufacturer's instruction. .. Primers were the following: FURIN-qF (forward primer): 5′-CAGAAGCATGGCTTCCACAAC-3′ FURIN-qR (reverse primer): 5′-TGTCACTGCTCTGTGCCAGAA-3′ ADAMTS1-qF (forward primer): 5′-CATAACAATGCTGCTATGTGCG-3′ ADAMTS1-qR (reverse primer): 5′-TGTCCGGCTGCAACTTCAG-3′ β -actin-qF: (forward primer): 5′-GGCTGTATTCCCCTCCATCG-3′ β -actin-qR: (reverse primer): 5′-CCAGTTGGTAACAATGCCATGT-3′.The experiments were repeated at least three times.

Article Title: Constitutive Expression of Yes-Associated Protein (Yap) in Adult Skeletal Muscle Fibres Induces Muscle Atrophy and Myopathy
Article Snippet: PCR primers for genotyping, end-point PCR primers for hYAP and Gapdh mRNA and qRT-PCR primers and probes (Roche Universal Probe Library). (DOCX) Click here for additional data file. .. PCR primers for genotyping, end-point PCR primers for hYAP and Gapdh mRNA and qRT-PCR primers and probes (Roche Universal Probe Library). (DOCX) Click here for additional data file.

Article Title: αII-spectrin and βII-spectrin do not affect TGFβ1-induced myofibroblast differentiation
Article Snippet: Paragraph title: RNA isolation, cDNA synthesis and qRT-PCR ... Gene expression quantification was performed using qRT-PCR analysis and SYBR Green Supermix (Roche, Basel, Switzerland).

Polymerase Chain Reaction:

Article Title: Constitutive Expression of Yes-Associated Protein (Yap) in Adult Skeletal Muscle Fibres Induces Muscle Atrophy and Myopathy
Article Snippet: Table S1 Primer details. .. PCR primers for genotyping, end-point PCR primers for hYAP and Gapdh mRNA and qRT-PCR primers and probes (Roche Universal Probe Library). (DOCX) Click here for additional data file. .. Video S1 Videos of MCK-tTA-hYAP1 S127A (hYAP1 S127A) and control mice following 5–7 weeks of doxycycline withdrawal.

Article Title: Circulating microRNAs -192 and -194 are associated with the presence and incidence of diabetes mellitus
Article Snippet: MiRNAs were measured using the ExiLENT SYBR Green Master Mix according to the manufacturer’s instructions. .. PCR assays were performed in triplicates on a LightCycler 480 II qRT-PCR System (Roche, Rotkreuz, Switzerland) and all miRNAs were detected at Cq < 37. .. The amplification curves were analyzed using the Light Cycler software version 1.5 (Roche).

Article Title: Interleukin-33 (IL-33) Increases Hyperoxia-Induced Bronchopulmonary Dysplasia in Newborn Mice by Regulation of Inflammatory Mediators
Article Snippet: The levels of IL-33, BCL-2, BAX, IL-1β, CXCL-1, MCP-1, TGF-β, and SMAD-7 mRNA were determined using SYBR Green and a LightCycler 480 quantitative real-time polymerase chain reaction (qRT-PCR) system (Roche). .. The levels of IL-33, BCL-2, BAX, IL-1β, CXCL-1, MCP-1, TGF-β, and SMAD-7 mRNA were determined using SYBR Green and a LightCycler 480 quantitative real-time polymerase chain reaction (qRT-PCR) system (Roche).

Injection:

Article Title: Production of Infectious Genotype 1b Virus Particles in Cell Culture and Impairment by Replication Enhancing Mutations
Article Snippet: Chimeric mice were inoculated by intraperitoneal injection of 100 µl of purified and concentrated culture supernatant (prepared as described above). .. EDTA plasma samples were collected at weekly intervals after inoculation and infection was monitored by a commercial qRT-PCR kit (Roche COBAS AmpliPrep/TaqMan48 assay, Roche Diagnostics).

Article Title: NLRP6, decreased in gastric cancer, suppresses tumorigenicity of gastric cancer cells
Article Snippet: Tumor xenografts were subjected to qRT-PCR analysis, TUNEL assay (Hoffman-La Roche Ltd), and IHC staining with anti-Ki-67 (Abcam). .. Tumor xenografts were subjected to qRT-PCR analysis, TUNEL assay (Hoffman-La Roche Ltd), and IHC staining with anti-Ki-67 (Abcam).

Binding Assay:

Article Title: Quantitative Real-Time PCR for Determination of Microcystin Synthetase E Copy Numbers for Microcystis and Anabaena in Lakes
Article Snippet: The QRT-PCR was performed with 1 μl of DNA from a standard strain or lake water sample, 3 mM MgCl2 , 0.5 μM concentrations of both primers (Sigma-Genosys, Ltd.), and 1 μl of hot start reaction mix to a final volume of 10 μl (LightCycler fastStart DNA master SYBR green I kit; Roche Diagnostics). .. Generation of the products was monitored after each extension step at 78°C in Microcystis and 77°C in Anabaena mcyE QRT-PCR by measuring the fluorescence of double-stranded DNA binding SYBR green 1 dye using LightCycler QRT-PCR (Roche Diagnostics). .. All lake water samples were amplified in triplicate.

Molecular Weight:

Article Title: Quantitative Real-Time PCR for Determination of Microcystin Synthetase E Copy Numbers for Microcystis and Anabaena in Lakes
Article Snippet: The mcyE copy numbers of the DNAs of the standard strains were calculated using the following equation assuming that each genome had only one mcyE gene and that the molecular weight of 1 bp was 660 g mol−1 : number of copies per microliter = (6 × 1023 )(DNA concentration)/molecular weight of one genome, where 6 × 1023 is the number of copies per mole, the DNA concentration is given in grams per microliter, and the molecular weight of one genome is given in grams per mole. .. Generation of the products was monitored after each extension step at 78°C in Microcystis and 77°C in Anabaena mcyE QRT-PCR by measuring the fluorescence of double-stranded DNA binding SYBR green 1 dye using LightCycler QRT-PCR (Roche Diagnostics).

In Vivo:

Article Title: NLRP6, decreased in gastric cancer, suppresses tumorigenicity of gastric cancer cells
Article Snippet: Paragraph title: In vivo tumorigenicity assay ... Tumor xenografts were subjected to qRT-PCR analysis, TUNEL assay (Hoffman-La Roche Ltd), and IHC staining with anti-Ki-67 (Abcam).

Fluorescence:

Article Title: Quantitative Real-Time PCR for Determination of Microcystin Synthetase E Copy Numbers for Microcystis and Anabaena in Lakes
Article Snippet: The QRT-PCR was performed with 1 μl of DNA from a standard strain or lake water sample, 3 mM MgCl2 , 0.5 μM concentrations of both primers (Sigma-Genosys, Ltd.), and 1 μl of hot start reaction mix to a final volume of 10 μl (LightCycler fastStart DNA master SYBR green I kit; Roche Diagnostics). .. Generation of the products was monitored after each extension step at 78°C in Microcystis and 77°C in Anabaena mcyE QRT-PCR by measuring the fluorescence of double-stranded DNA binding SYBR green 1 dye using LightCycler QRT-PCR (Roche Diagnostics). .. All lake water samples were amplified in triplicate.

Tumorigenicity Assay:

Article Title: NLRP6, decreased in gastric cancer, suppresses tumorigenicity of gastric cancer cells
Article Snippet: Paragraph title: In vivo tumorigenicity assay ... Tumor xenografts were subjected to qRT-PCR analysis, TUNEL assay (Hoffman-La Roche Ltd), and IHC staining with anti-Ki-67 (Abcam).

Immunohistochemistry:

Article Title: NLRP6, decreased in gastric cancer, suppresses tumorigenicity of gastric cancer cells
Article Snippet: At 27 days after cell inoculation, the mice were sacrificed and the tumors were collected. .. Tumor xenografts were subjected to qRT-PCR analysis, TUNEL assay (Hoffman-La Roche Ltd), and IHC staining with anti-Ki-67 (Abcam). .. To evaluate the cell migration and invasive capacity of cells, Transwell assays were performed using Matrigel noncoated and Matrigel-coated Boyden chamber (BD Biosciences), respectively.

Mouse Assay:

Article Title: Production of Infectious Genotype 1b Virus Particles in Cell Culture and Impairment by Replication Enhancing Mutations
Article Snippet: Paragraph title: Infection of uPA+/+-SCID mice ... EDTA plasma samples were collected at weekly intervals after inoculation and infection was monitored by a commercial qRT-PCR kit (Roche COBAS AmpliPrep/TaqMan48 assay, Roche Diagnostics).

Article Title: NLRP6, decreased in gastric cancer, suppresses tumorigenicity of gastric cancer cells
Article Snippet: At 27 days after cell inoculation, the mice were sacrificed and the tumors were collected. .. Tumor xenografts were subjected to qRT-PCR analysis, TUNEL assay (Hoffman-La Roche Ltd), and IHC staining with anti-Ki-67 (Abcam).

Article Title: Constitutive Expression of Yes-Associated Protein (Yap) in Adult Skeletal Muscle Fibres Induces Muscle Atrophy and Myopathy
Article Snippet: Values present mean ±SEM and displayed as fold change relative to control mice (n = 4–8). (TIF) Click here for additional data file. .. PCR primers for genotyping, end-point PCR primers for hYAP and Gapdh mRNA and qRT-PCR primers and probes (Roche Universal Probe Library). (DOCX) Click here for additional data file.

Reverse Transcription Polymerase Chain Reaction:

Article Title: Involvement of the Interleukin-23/Interleukin-17 Axis in Chronic Hepatitis C Virus Infection and Its Treatment Responses
Article Snippet: Serum HCV antibody was detected by enzyme-linked immunosorbent assay (ELISA) using a commercial detection kit (Livzon Diagnostics Inc., Zhuhai, China). .. Plasma HCV RNA load was determined using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay (Cobas Taqman HCV Test, Roche Diagnostics, Indianapolis, IN, USA). .. With this method, the lowest detection limit is 15 IU/mL.

Periodic Counter-current Chromatography:

Article Title: Quantitative Real-Time PCR for Determination of Microcystin Synthetase E Copy Numbers for Microcystis and Anabaena in Lakes
Article Snippet: These genome sizes were estimated on the basis of the genome sizes of Microcystis sp. strain PCC 7941, Anabaena sp. strain PCC 6309, and Anabaena sp. strain PCC 7122 ( ). .. Generation of the products was monitored after each extension step at 78°C in Microcystis and 77°C in Anabaena mcyE QRT-PCR by measuring the fluorescence of double-stranded DNA binding SYBR green 1 dye using LightCycler QRT-PCR (Roche Diagnostics).

Purification:

Article Title: The novel lncRNA lnc-NR2F1 is pro-neurogenic and mutated in human neurodevelopmental disorders
Article Snippet: Isolated RNA was subjected to DNAse treatment using TurboDNase and purified by phenol-chloroform extraction and ethanol precipitation. .. For qRT-PCR analysis we used Roche’s Lightcycler and Stratagene’s RT kit.

Article Title: Genome-wide identification and characterization of miRNAome from tomato (Solanum lycopersicum) roots and root-knot nematode (Meloidogyne incognita) during susceptible interaction
Article Snippet: The polyadenylated product was purified and reverse transcribed using a poly-T adapter and SuperScript Reverse Transcriptase III (Invitrogen). .. Further, expression of miRNAs was measured through Taqman probe based qRT- PCR method (Roche, Mannheim, Germany).

Article Title: Production of Infectious Genotype 1b Virus Particles in Cell Culture and Impairment by Replication Enhancing Mutations
Article Snippet: Chimeric mice were inoculated by intraperitoneal injection of 100 µl of purified and concentrated culture supernatant (prepared as described above). .. EDTA plasma samples were collected at weekly intervals after inoculation and infection was monitored by a commercial qRT-PCR kit (Roche COBAS AmpliPrep/TaqMan48 assay, Roche Diagnostics).

Article Title: αII-spectrin and βII-spectrin do not affect TGFβ1-induced myofibroblast differentiation
Article Snippet: To obtain total RNA, the FavorPrep Tissue Total RNA Purification Mini Kit (FATRK; Favorgen Biotech Corp., Taiwan) was used in accordance with the manufacturer’s protocol. .. Gene expression quantification was performed using qRT-PCR analysis and SYBR Green Supermix (Roche, Basel, Switzerland).

RNA Extraction:

Article Title: The novel lncRNA lnc-NR2F1 is pro-neurogenic and mutated in human neurodevelopmental disorders
Article Snippet: Proteinase K treatment proceeded for 45 min at 45C, followed by RNA extraction using Trizol. .. For qRT-PCR analysis we used Roche’s Lightcycler and Stratagene’s RT kit.

Article Title: Interleukin-33 (IL-33) Increases Hyperoxia-Induced Bronchopulmonary Dysplasia in Newborn Mice by Regulation of Inflammatory Mediators
Article Snippet: Paragraph title: RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR) ... The levels of IL-33, BCL-2, BAX, IL-1β, CXCL-1, MCP-1, TGF-β, and SMAD-7 mRNA were determined using SYBR Green and a LightCycler 480 quantitative real-time polymerase chain reaction (qRT-PCR) system (Roche).

Software:

Article Title: Quantitative Real-Time PCR for Determination of Microcystin Synthetase E Copy Numbers for Microcystis and Anabaena in Lakes
Article Snippet: Generation of the products was monitored after each extension step at 78°C in Microcystis and 77°C in Anabaena mcyE QRT-PCR by measuring the fluorescence of double-stranded DNA binding SYBR green 1 dye using LightCycler QRT-PCR (Roche Diagnostics). .. Generation of the products was monitored after each extension step at 78°C in Microcystis and 77°C in Anabaena mcyE QRT-PCR by measuring the fluorescence of double-stranded DNA binding SYBR green 1 dye using LightCycler QRT-PCR (Roche Diagnostics).

Article Title: Time-Dependent Effects of POT1 Knockdown on Proliferation, Tumorigenicity, and HDACi Response of SK-OV3 Ovarian Cancer Cells
Article Snippet: The primers indicated below, which were specific for hTERT mRNA and the TaqMan probe [ ], were also used in the qRT-PCR assays (Roche, Indianapolis, IN, USA): forward 5′-TGACACCTCACCTCACCCAC-3′ and reverse 5′-CACTGTCTTCCGCAAGTTCAC-3′ and TaqMan probe 5′-ACCCTGGTCCGAGGTGTCCCTGAG-3′. .. The relative expression levels of the target genes were estimated using the ΔΔCT method.

Article Title: Circulating microRNAs -192 and -194 are associated with the presence and incidence of diabetes mellitus
Article Snippet: The amplification curves were examined with the SDS 2.4 software (Applied Biosystems). .. PCR assays were performed in triplicates on a LightCycler 480 II qRT-PCR System (Roche, Rotkreuz, Switzerland) and all miRNAs were detected at Cq < 37.

Article Title: αII-spectrin and βII-spectrin do not affect TGFβ1-induced myofibroblast differentiation
Article Snippet: Gene expression quantification was performed using qRT-PCR analysis and SYBR Green Supermix (Roche, Basel, Switzerland). .. Gene expression quantification was performed using qRT-PCR analysis and SYBR Green Supermix (Roche, Basel, Switzerland).

Real-time Polymerase Chain Reaction:

Article Title: Oocyte-specific deletion of furin leads to female infertility by causing early secondary follicle arrest in mice
Article Snippet: Paragraph title: Quantitative real time-PCR ... RNA was isolated from ~100 oocytes for each group and reverse transcription reaction was carried out using the RNeasy Kit (Qiagen, Hilden, Germany). mRNA level of each gene was validated by qRT-PCR analysis (Roche; 480, Boehringer, Mannheim, Germany) according to the manufacturer's instruction.

Article Title: Time-Dependent Effects of POT1 Knockdown on Proliferation, Tumorigenicity, and HDACi Response of SK-OV3 Ovarian Cancer Cells
Article Snippet: Paragraph title: 2.3. Quantitative Real-Time PCR (qRT-PCR) ... The primers indicated below, which were specific for hTERT mRNA and the TaqMan probe [ ], were also used in the qRT-PCR assays (Roche, Indianapolis, IN, USA): forward 5′-TGACACCTCACCTCACCCAC-3′ and reverse 5′-CACTGTCTTCCGCAAGTTCAC-3′ and TaqMan probe 5′-ACCCTGGTCCGAGGTGTCCCTGAG-3′.

Article Title: Carnosine Prevents Aβ-Induced Oxidative Stress and Inflammation in Microglial Cells: A Key Role of TGF-β1
Article Snippet: Paragraph title: 2.7. Gene Expression Analysis by Quantitative Real-Time PCR (qRT-PCR) ... Next, each sample was quantified, diluted to a final concentration of 25 ng/µL, and used for qRT-PCR analysis (LightCycler® 480 System, Roche Molecular Systems, Inc., Pleasanton, CA, USA).

Article Title: The Utilization of RNA Silencing Technology to Mitigate the Voriconazole Resistance of Aspergillus Flavus; Lipofectamine-Based Delivery
Article Snippet: Paragraph title: Quantitative Real-Time PCR (qRT-PCR) ... Cyp51A and MDR1 mRNA levels were amplified by qRT-PCR instrument (light cycler 96, Roche) using SYBR Green Master Mix (AMPLIQON, Denmark).

Article Title: αII-spectrin and βII-spectrin do not affect TGFβ1-induced myofibroblast differentiation
Article Snippet: Gene expression quantification was performed using qRT-PCR analysis and SYBR Green Supermix (Roche, Basel, Switzerland). .. Gene expression quantification was performed using qRT-PCR analysis and SYBR Green Supermix (Roche, Basel, Switzerland).

Article Title: Interleukin-33 (IL-33) Increases Hyperoxia-Induced Bronchopulmonary Dysplasia in Newborn Mice by Regulation of Inflammatory Mediators
Article Snippet: Total RNA was extracted from cells of the lungs from the three mouse groups using Trizol reagent. .. The levels of IL-33, BCL-2, BAX, IL-1β, CXCL-1, MCP-1, TGF-β, and SMAD-7 mRNA were determined using SYBR Green and a LightCycler 480 quantitative real-time polymerase chain reaction (qRT-PCR) system (Roche). .. GAPDH was used as a control.

Negative Control:

Article Title: Time-Dependent Effects of POT1 Knockdown on Proliferation, Tumorigenicity, and HDACi Response of SK-OV3 Ovarian Cancer Cells
Article Snippet: Nuclease-free water was used as a negative control, and β -2M was used as an internal control. .. The primers indicated below, which were specific for hTERT mRNA and the TaqMan probe [ ], were also used in the qRT-PCR assays (Roche, Indianapolis, IN, USA): forward 5′-TGACACCTCACCTCACCCAC-3′ and reverse 5′-CACTGTCTTCCGCAAGTTCAC-3′ and TaqMan probe 5′-ACCCTGGTCCGAGGTGTCCCTGAG-3′.

Article Title: Carnosine Prevents Aβ-Induced Oxidative Stress and Inflammation in Microglial Cells: A Key Role of TGF-β1
Article Snippet: Next, each sample was quantified, diluted to a final concentration of 25 ng/µL, and used for qRT-PCR analysis (LightCycler® 480 System, Roche Molecular Systems, Inc., Pleasanton, CA, USA). .. Next, each sample was quantified, diluted to a final concentration of 25 ng/µL, and used for qRT-PCR analysis (LightCycler® 480 System, Roche Molecular Systems, Inc., Pleasanton, CA, USA).

RNA Expression:

Article Title: Carnosine Prevents Aβ-Induced Oxidative Stress and Inflammation in Microglial Cells: A Key Role of TGF-β1
Article Snippet: Next, each sample was quantified, diluted to a final concentration of 25 ng/µL, and used for qRT-PCR analysis (LightCycler® 480 System, Roche Molecular Systems, Inc., Pleasanton, CA, USA). .. As a negative control, a reaction in the absence of cDNA (no template control, NTC) was performed and verified by using an Agilent Bioanalyzer 2100 with Agilent DNA 1000 Kit.

Transgenic Assay:

Article Title: Production of Infectious Genotype 1b Virus Particles in Cell Culture and Impairment by Replication Enhancing Mutations
Article Snippet: Transgenic SCID mice overexpressing the uPA gene under the control of an albumin promoter (uPA+/+ -SCID) were xenografted with primary human hepatocytes as described elsewhere . .. EDTA plasma samples were collected at weekly intervals after inoculation and infection was monitored by a commercial qRT-PCR kit (Roche COBAS AmpliPrep/TaqMan48 assay, Roche Diagnostics).

Article Title: Constitutive Expression of Yes-Associated Protein (Yap) in Adult Skeletal Muscle Fibres Induces Muscle Atrophy and Myopathy
Article Snippet: TA muscles of transgenic mice were harvested 3 weeks or 5–7 weeks following doxycycline withdrawal. .. PCR primers for genotyping, end-point PCR primers for hYAP and Gapdh mRNA and qRT-PCR primers and probes (Roche Universal Probe Library). (DOCX) Click here for additional data file.

Enzyme-linked Immunosorbent Assay:

Article Title: Involvement of the Interleukin-23/Interleukin-17 Axis in Chronic Hepatitis C Virus Infection and Its Treatment Responses
Article Snippet: Serum HCV antibody was detected by enzyme-linked immunosorbent assay (ELISA) using a commercial detection kit (Livzon Diagnostics Inc., Zhuhai, China). .. Plasma HCV RNA load was determined using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay (Cobas Taqman HCV Test, Roche Diagnostics, Indianapolis, IN, USA).

Ethanol Precipitation:

Article Title: The novel lncRNA lnc-NR2F1 is pro-neurogenic and mutated in human neurodevelopmental disorders
Article Snippet: Isolated RNA was subjected to DNAse treatment using TurboDNase and purified by phenol-chloroform extraction and ethanol precipitation. .. For qRT-PCR analysis we used Roche’s Lightcycler and Stratagene’s RT kit.

Spectrophotometry:

Article Title: Carnosine Prevents Aβ-Induced Oxidative Stress and Inflammation in Microglial Cells: A Key Role of TGF-β1
Article Snippet: The concentration of total RNA recovered from 3.5 × 105 cells (previously seeded in 12-well plates) treated for 6 h was determined by measuring the absorbance at 260 nm with a Varioskan® Flash spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). .. Next, each sample was quantified, diluted to a final concentration of 25 ng/µL, and used for qRT-PCR analysis (LightCycler® 480 System, Roche Molecular Systems, Inc., Pleasanton, CA, USA).

Article Title: αII-spectrin and βII-spectrin do not affect TGFβ1-induced myofibroblast differentiation
Article Snippet: RNA concentration and purity were determined by UV spectrophotometry (NanoDrop Technologies, Wilmington, NC). .. Gene expression quantification was performed using qRT-PCR analysis and SYBR Green Supermix (Roche, Basel, Switzerland).

Activation Assay:

Article Title: Genome-wide identification and characterization of miRNAome from tomato (Solanum lycopersicum) roots and root-knot nematode (Meloidogyne incognita) during susceptible interaction
Article Snippet: Further, expression of miRNAs was measured through Taqman probe based qRT- PCR method (Roche, Mannheim, Germany). .. Further, expression of miRNAs was measured through Taqman probe based qRT- PCR method (Roche, Mannheim, Germany).

Staining:

Article Title: NLRP6, decreased in gastric cancer, suppresses tumorigenicity of gastric cancer cells
Article Snippet: At 27 days after cell inoculation, the mice were sacrificed and the tumors were collected. .. Tumor xenografts were subjected to qRT-PCR analysis, TUNEL assay (Hoffman-La Roche Ltd), and IHC staining with anti-Ki-67 (Abcam). .. To evaluate the cell migration and invasive capacity of cells, Transwell assays were performed using Matrigel noncoated and Matrigel-coated Boyden chamber (BD Biosciences), respectively.

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    Roche lightcycler 480 qrt pcr system
    <t>qRT-PCR</t> analysis of tissue-specific relative expression levels of locust insulin-related peptide-like 1 ( LIRP ) across castes of B. terrestris . LIRP is part of the insulin signaling involved in reproduction and diapause. It is most abundant in fat body and ventriculus of males and reproducing queens (see main text). Data represent mean ± SEM ( n = 3–5) of normalized and rescaled expression levels. (A) Heatmap: Log-scale. Bar graphs: Relative transcript levels in linear scale (B) and log-transformed scale (C) . Significantly different expression levels are indicated by different letters ( p
    Lightcycler 480 Qrt Pcr System, supplied by Roche, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lightcycler 480 qrt pcr system/product/Roche
    Average 91 stars, based on 3 article reviews
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    Roche qrt pcr lightcycler 96 instrument
    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for <t>qRT-PCR</t> assays, utilizing the GeNorm algorithm, for ( A ) normal human Chondrocytes (hChondrocytes) and ( B ) hChondrocytes undergoing apoptosis or ( C ) both normal and apoptotic chondrocyte cell lines combined.
    Qrt Pcr Lightcycler 96 Instrument, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche sybr green based qrt pcr
    FOXO1 represses T-bet and miR-31 in Th1 cells. (A) Correlation between miR-31 expression normalized to snU6 and Foxo1 expression normalized to Hprt determined five times in 6 day intervals from naive to Th1 rep cells <t>(qRT-PCR)</t> ( n = 15 from one experiment, p value is depicted in the figure). (B) Foxo1 , Foxo3 and pri-miR-31 expression normalized to Hprt in repeatedly (two rounds of stimulation) activated Th1 cells, treated with a pool of 8 siRNAs specific for Foxo1 and Foxo3 or a SI-SCR control, analyzed 48 h after siRNA treatment by qRT-PCR, presented relative to the SI-SCR control. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, * p ≤ 0.05, ** p ≤ 0.01) (C) Klf2, Sell, Cd69 and pri-miR-31 expression normalized to Hprt in activated CD4 + cells transduced 36–40 h post activation with a retroviral vector expressing a constitutive active FOXO1 (FOXO1A3) or an empty control vector (RV). Cells were cultured under Th1 polarizing conditions for additional 48 h. Expression was analyzed by qRT-PCR 48 h post transduction and is presented relative to RV. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01) (D) Representative intracellular protein staining and T-Bet protein expression in the samples analyzed in (C) , presented as MFI of T-Bet, normalized to RV assessed by flow cytometry. Data is shown as mean +SEM, n = 5–11 pooled from three independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01). (E) Foxo1 expression normalized to Hprt in Th1 rep cells activated with αCD3/28 under Th1 polarizing conditions for 48 h ±TGFβ, presented relative to values obtained from untreated Th1 rep cells determined by qRT-PCR. Data is shown as mean +SEM, n = 9 pooled from 3 independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01, *** p ≤ 0.001). MiR-31 expression normalized to snU6 in the same cells, presented relative to Th1 rep cells before reactivation assessed by qRT-PCR. Data is shown as mean +SEM, n = 9 pooled from three independent experiments (One-way Anova with Dunn's test for multiple comparison, * p ≤ 0.05, *** p ≤ 0.001). (F) Representative intracellular protein staining of Th1 rep cells activated with αCD3/28 under Th1 polarizing conditions for 48 h ±TGFβ and T-Bet protein expression, presented as MFI of T-Bet, normalized to untreated Th1 rep cells. Data is shown as mean +SEM, n = 2–3 pooled from four independent experiments (Mann-Whitney test for unpaired data, *** p ≤ 0.001). Ifng expression normalized to Hprt in the samples analyzed in (A) , presented relative to RV. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, * p ≤ 0.05, ** p ≤ 0.01). (G) Representative intracellular FOXP3 protein staining of Th1 rep cells activated with αCD3/28 in Th1 polarizing conditions for 48 h ±TGF-β.
    Sybr Green Based Qrt Pcr, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    qRT-PCR analysis of tissue-specific relative expression levels of locust insulin-related peptide-like 1 ( LIRP ) across castes of B. terrestris . LIRP is part of the insulin signaling involved in reproduction and diapause. It is most abundant in fat body and ventriculus of males and reproducing queens (see main text). Data represent mean ± SEM ( n = 3–5) of normalized and rescaled expression levels. (A) Heatmap: Log-scale. Bar graphs: Relative transcript levels in linear scale (B) and log-transformed scale (C) . Significantly different expression levels are indicated by different letters ( p

    Journal: Frontiers in Physiology

    Article Title: Gene Expression Dynamics in Major Endocrine Regulatory Pathways along the Transition from Solitary to Social Life in a Bumblebee, Bombus terrestris

    doi: 10.3389/fphys.2016.00574

    Figure Lengend Snippet: qRT-PCR analysis of tissue-specific relative expression levels of locust insulin-related peptide-like 1 ( LIRP ) across castes of B. terrestris . LIRP is part of the insulin signaling involved in reproduction and diapause. It is most abundant in fat body and ventriculus of males and reproducing queens (see main text). Data represent mean ± SEM ( n = 3–5) of normalized and rescaled expression levels. (A) Heatmap: Log-scale. Bar graphs: Relative transcript levels in linear scale (B) and log-transformed scale (C) . Significantly different expression levels are indicated by different letters ( p

    Article Snippet: We performed qRT-PCR using a LightCycler 480 qRT-PCR System (Roche) with SYBR green fluorescent labels and 200 nM of each primer.

    Techniques: Quantitative RT-PCR, Expressing, Transformation Assay

    qRT-PCR analysis of tissue-specific relative expression levels of forkhead box protein O ( FOXO ) across castes of B. terrestris . FOXO is a transcription factor involved in stress tolerance, diapause, longevity, and growth. FOXO transcription is lower in workers and reproducing queens than in other castes (see main text). Data represent mean ± SEM ( n = 3–5) of normalized and rescaled expression levels. (A) Heatmap: Log-scale. Bar graphs: Relative transcript levels in linear scale (B) and log-transformed scale (C) . Significantly different expression levels are indicated by different letters ( p

    Journal: Frontiers in Physiology

    Article Title: Gene Expression Dynamics in Major Endocrine Regulatory Pathways along the Transition from Solitary to Social Life in a Bumblebee, Bombus terrestris

    doi: 10.3389/fphys.2016.00574

    Figure Lengend Snippet: qRT-PCR analysis of tissue-specific relative expression levels of forkhead box protein O ( FOXO ) across castes of B. terrestris . FOXO is a transcription factor involved in stress tolerance, diapause, longevity, and growth. FOXO transcription is lower in workers and reproducing queens than in other castes (see main text). Data represent mean ± SEM ( n = 3–5) of normalized and rescaled expression levels. (A) Heatmap: Log-scale. Bar graphs: Relative transcript levels in linear scale (B) and log-transformed scale (C) . Significantly different expression levels are indicated by different letters ( p

    Article Snippet: We performed qRT-PCR using a LightCycler 480 qRT-PCR System (Roche) with SYBR green fluorescent labels and 200 nM of each primer.

    Techniques: Quantitative RT-PCR, Expressing, Transformation Assay

    qRT-PCR analysis of tissue-specific relative expression levels of insulin-like peptide receptor 2 ( InR-2 ) across castes of B. terrestris . InR-2 is a receptor involved in insulin signaling. It is upregulated in gonads (see main text). Data represent mean ± SEM ( n = 3–5) of normalized and rescaled expression levels. (A) Heatmap: Log-scale. Bar graphs: Relative transcript levels in linear scale (B) and log-transformed scale (C) . Significantly different expression levels are indicated by different letters ( p

    Journal: Frontiers in Physiology

    Article Title: Gene Expression Dynamics in Major Endocrine Regulatory Pathways along the Transition from Solitary to Social Life in a Bumblebee, Bombus terrestris

    doi: 10.3389/fphys.2016.00574

    Figure Lengend Snippet: qRT-PCR analysis of tissue-specific relative expression levels of insulin-like peptide receptor 2 ( InR-2 ) across castes of B. terrestris . InR-2 is a receptor involved in insulin signaling. It is upregulated in gonads (see main text). Data represent mean ± SEM ( n = 3–5) of normalized and rescaled expression levels. (A) Heatmap: Log-scale. Bar graphs: Relative transcript levels in linear scale (B) and log-transformed scale (C) . Significantly different expression levels are indicated by different letters ( p

    Article Snippet: We performed qRT-PCR using a LightCycler 480 qRT-PCR System (Roche) with SYBR green fluorescent labels and 200 nM of each primer.

    Techniques: Quantitative RT-PCR, Expressing, Transformation Assay

    qRT-PCR analysis of tissue-specific relative expression levels of adipokinetic hormone ( AKH ) across castes of B. terrestris . AKH is involved in the mobilization of lipid, carbohydrates and/or proline stores. AKH is mainly expressed in brain-CC-CA samples, but minor expression occurred also in other tissues (see main text). Data represent mean ± SEM ( n = 3–5) of normalized and rescaled expression levels. (A) Heatmap: Log-scale. Bar graphs: Relative transcript levels in linear scale (B) and log-transformed scale (C) . Significantly different expression levels are indicated by different letters ( p

    Journal: Frontiers in Physiology

    Article Title: Gene Expression Dynamics in Major Endocrine Regulatory Pathways along the Transition from Solitary to Social Life in a Bumblebee, Bombus terrestris

    doi: 10.3389/fphys.2016.00574

    Figure Lengend Snippet: qRT-PCR analysis of tissue-specific relative expression levels of adipokinetic hormone ( AKH ) across castes of B. terrestris . AKH is involved in the mobilization of lipid, carbohydrates and/or proline stores. AKH is mainly expressed in brain-CC-CA samples, but minor expression occurred also in other tissues (see main text). Data represent mean ± SEM ( n = 3–5) of normalized and rescaled expression levels. (A) Heatmap: Log-scale. Bar graphs: Relative transcript levels in linear scale (B) and log-transformed scale (C) . Significantly different expression levels are indicated by different letters ( p

    Article Snippet: We performed qRT-PCR using a LightCycler 480 qRT-PCR System (Roche) with SYBR green fluorescent labels and 200 nM of each primer.

    Techniques: Quantitative RT-PCR, Expressing, Transformation Assay

    qRT-PCR analysis of tissue-specific relative expression levels of Krüppel homolog 1 ( Kr-h1 ) across castes of B. terrestris . Kr-h1 is a transcription factor downstream of juvenile hormone receptors. It is downregulated in virgin queens, and upregulated in males and diapausing queens (see main text). Data represent mean ± SEM ( n = 3–5) of normalized and rescaled expression levels. (A) Heatmap: Log-scale. Bar graphs: Relative transcript levels in linear scale (B) and log-transformed scale (C) . Significantly different expression levels are indicated by different letters ( p

    Journal: Frontiers in Physiology

    Article Title: Gene Expression Dynamics in Major Endocrine Regulatory Pathways along the Transition from Solitary to Social Life in a Bumblebee, Bombus terrestris

    doi: 10.3389/fphys.2016.00574

    Figure Lengend Snippet: qRT-PCR analysis of tissue-specific relative expression levels of Krüppel homolog 1 ( Kr-h1 ) across castes of B. terrestris . Kr-h1 is a transcription factor downstream of juvenile hormone receptors. It is downregulated in virgin queens, and upregulated in males and diapausing queens (see main text). Data represent mean ± SEM ( n = 3–5) of normalized and rescaled expression levels. (A) Heatmap: Log-scale. Bar graphs: Relative transcript levels in linear scale (B) and log-transformed scale (C) . Significantly different expression levels are indicated by different letters ( p

    Article Snippet: We performed qRT-PCR using a LightCycler 480 qRT-PCR System (Roche) with SYBR green fluorescent labels and 200 nM of each primer.

    Techniques: Quantitative RT-PCR, Expressing, Transformation Assay

    qRT-PCR analysis of tissue-specific relative expression levels of vitellogenin ( Vg ) across castes of B. terrestris . Vg is a yolk protein with multiple functions in immunity and behavioral regulation. It is highly upregulated in fat body of all castes, and generally lowest in males and diapausing queens (see main text). Data represent mean ± SEM ( n = 3–5) of normalized and rescaled expression levels. (A) Heatmap: Log-scale. Bar graphs: Relative transcript levels in linear scale (B) and log-transformed scale (C) . Significantly different expression levels are indicated by different letters ( p

    Journal: Frontiers in Physiology

    Article Title: Gene Expression Dynamics in Major Endocrine Regulatory Pathways along the Transition from Solitary to Social Life in a Bumblebee, Bombus terrestris

    doi: 10.3389/fphys.2016.00574

    Figure Lengend Snippet: qRT-PCR analysis of tissue-specific relative expression levels of vitellogenin ( Vg ) across castes of B. terrestris . Vg is a yolk protein with multiple functions in immunity and behavioral regulation. It is highly upregulated in fat body of all castes, and generally lowest in males and diapausing queens (see main text). Data represent mean ± SEM ( n = 3–5) of normalized and rescaled expression levels. (A) Heatmap: Log-scale. Bar graphs: Relative transcript levels in linear scale (B) and log-transformed scale (C) . Significantly different expression levels are indicated by different letters ( p

    Article Snippet: We performed qRT-PCR using a LightCycler 480 qRT-PCR System (Roche) with SYBR green fluorescent labels and 200 nM of each primer.

    Techniques: Quantitative RT-PCR, Expressing, Transformation Assay

    qRT-PCR analysis of tissue-specific relative expression levels of insulin-like growth factor 1 ( IGF-1 ) across castes of B. terrestris . IGF-1 is part of the IIS signaling involved in reproduction and diapause. It is queen-specific (i.e., absent in workers and males) and lowest in virgin queens (see main text). Data represent mean ± SEM ( n = 3–5) of normalized and rescaled expression levels. (A) Heatmap: Log-scale. Bar graphs: Relative transcript levels in (B) linear scale; and (C) log-transformed scale. Significantly different expression levels are indicated by different letters ( p

    Journal: Frontiers in Physiology

    Article Title: Gene Expression Dynamics in Major Endocrine Regulatory Pathways along the Transition from Solitary to Social Life in a Bumblebee, Bombus terrestris

    doi: 10.3389/fphys.2016.00574

    Figure Lengend Snippet: qRT-PCR analysis of tissue-specific relative expression levels of insulin-like growth factor 1 ( IGF-1 ) across castes of B. terrestris . IGF-1 is part of the IIS signaling involved in reproduction and diapause. It is queen-specific (i.e., absent in workers and males) and lowest in virgin queens (see main text). Data represent mean ± SEM ( n = 3–5) of normalized and rescaled expression levels. (A) Heatmap: Log-scale. Bar graphs: Relative transcript levels in (B) linear scale; and (C) log-transformed scale. Significantly different expression levels are indicated by different letters ( p

    Article Snippet: We performed qRT-PCR using a LightCycler 480 qRT-PCR System (Roche) with SYBR green fluorescent labels and 200 nM of each primer.

    Techniques: Quantitative RT-PCR, Expressing, Transformation Assay

    qRT-PCR analysis of tissue-specific relative expression levels of insulin-like peptide receptor 1 ( InR-1 ) across castes of B. terrestris . InR-1 is a receptor involved in insulin signaling. It is upregulated in virgin and diapausing queens, especially in crop and hypopharyngeal glands (see main text). Data represent mean ± SEM ( n = 3–5) of normalized and rescaled expression levels. (A) Heatmap: Log-scale. Bar graphs: Relative transcript levels in linear scale (B) and log-transformed scale (C) . Significantly different expression levels are indicated by different letters ( p

    Journal: Frontiers in Physiology

    Article Title: Gene Expression Dynamics in Major Endocrine Regulatory Pathways along the Transition from Solitary to Social Life in a Bumblebee, Bombus terrestris

    doi: 10.3389/fphys.2016.00574

    Figure Lengend Snippet: qRT-PCR analysis of tissue-specific relative expression levels of insulin-like peptide receptor 1 ( InR-1 ) across castes of B. terrestris . InR-1 is a receptor involved in insulin signaling. It is upregulated in virgin and diapausing queens, especially in crop and hypopharyngeal glands (see main text). Data represent mean ± SEM ( n = 3–5) of normalized and rescaled expression levels. (A) Heatmap: Log-scale. Bar graphs: Relative transcript levels in linear scale (B) and log-transformed scale (C) . Significantly different expression levels are indicated by different letters ( p

    Article Snippet: We performed qRT-PCR using a LightCycler 480 qRT-PCR System (Roche) with SYBR green fluorescent labels and 200 nM of each primer.

    Techniques: Quantitative RT-PCR, Expressing, Transformation Assay

    qRT-PCR analysis of tissue-specific relative expression levels of methyl farnesoate epoxidase ( MFE ) across castes of B. terrestris . MFE is crucial in synthetizing juvenile hormone (JH). It is heavily upregulated in gonads of workers and reproducing queen. It is also found in brains of all samples (see main text). Data represent mean ± SEM ( n = 3–5) of normalized and rescaled expression levels. (A) Heatmap: Log-scale. Bar graphs: Relative transcript levels in linear scale (B) and log-transformed scale (C) . Significantly different expression levels are indicated by different letters ( p

    Journal: Frontiers in Physiology

    Article Title: Gene Expression Dynamics in Major Endocrine Regulatory Pathways along the Transition from Solitary to Social Life in a Bumblebee, Bombus terrestris

    doi: 10.3389/fphys.2016.00574

    Figure Lengend Snippet: qRT-PCR analysis of tissue-specific relative expression levels of methyl farnesoate epoxidase ( MFE ) across castes of B. terrestris . MFE is crucial in synthetizing juvenile hormone (JH). It is heavily upregulated in gonads of workers and reproducing queen. It is also found in brains of all samples (see main text). Data represent mean ± SEM ( n = 3–5) of normalized and rescaled expression levels. (A) Heatmap: Log-scale. Bar graphs: Relative transcript levels in linear scale (B) and log-transformed scale (C) . Significantly different expression levels are indicated by different letters ( p

    Article Snippet: We performed qRT-PCR using a LightCycler 480 qRT-PCR System (Roche) with SYBR green fluorescent labels and 200 nM of each primer.

    Techniques: Quantitative RT-PCR, Expressing, Transformation Assay

    qRT-PCR analysis of tissue-specific relative expression levels of adipokinetic hormone receptor ( AKHR ) across castes of B. terrestris . AKHR is involved in the regulation of energy storage. It is mainly expressed in fat body and hypopharyngeal glands (see main text). Data represent mean ± SEM ( n = 3–5) of normalized and rescaled expression levels. (A) Heatmap: Log-scale. Bar graphs: relative transcript levels in linear scale (B) and log-transformed scale (C) . Significantly different expression levels are indicated by different letters ( p

    Journal: Frontiers in Physiology

    Article Title: Gene Expression Dynamics in Major Endocrine Regulatory Pathways along the Transition from Solitary to Social Life in a Bumblebee, Bombus terrestris

    doi: 10.3389/fphys.2016.00574

    Figure Lengend Snippet: qRT-PCR analysis of tissue-specific relative expression levels of adipokinetic hormone receptor ( AKHR ) across castes of B. terrestris . AKHR is involved in the regulation of energy storage. It is mainly expressed in fat body and hypopharyngeal glands (see main text). Data represent mean ± SEM ( n = 3–5) of normalized and rescaled expression levels. (A) Heatmap: Log-scale. Bar graphs: relative transcript levels in linear scale (B) and log-transformed scale (C) . Significantly different expression levels are indicated by different letters ( p

    Article Snippet: We performed qRT-PCR using a LightCycler 480 qRT-PCR System (Roche) with SYBR green fluorescent labels and 200 nM of each primer.

    Techniques: Quantitative RT-PCR, Expressing, Transformation Assay

    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal human Chondrocytes (hChondrocytes) and ( B ) hChondrocytes undergoing apoptosis or ( C ) both normal and apoptotic chondrocyte cell lines combined.

    Journal: Scientific Reports

    Article Title: Recommendations for improving accuracy of gene expression data in bone and cartilage tissue engineering

    doi: 10.1038/s41598-018-33242-z

    Figure Lengend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal human Chondrocytes (hChondrocytes) and ( B ) hChondrocytes undergoing apoptosis or ( C ) both normal and apoptotic chondrocyte cell lines combined.

    Article Snippet: The PCR reactions were performed using a qRT-PCR LightCycler® 96 Instrument (Roche, Basel, Swiss), where the total volume per reaction was 10 μl, containing 10 μM of each reference primer (Table ), the corresponding diluted cDNA and 2x FastStart Essential DNA Green Master (Roche).

    Techniques: Expressing, Quantitative RT-PCR

    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm. ( A ) GeNorm results between Control cell groups with normal hChondrocytes, hBMSCs and hADSCs. ( B ) GeNorm results between treated cell groups, i.e. hChondrocytes undergoing apoptosis, chondrogenic differentiated hBMSCs and hADSCs. ( C ) GeNorm results between all untreated and treated cell lines and types.

    Journal: Scientific Reports

    Article Title: Recommendations for improving accuracy of gene expression data in bone and cartilage tissue engineering

    doi: 10.1038/s41598-018-33242-z

    Figure Lengend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm. ( A ) GeNorm results between Control cell groups with normal hChondrocytes, hBMSCs and hADSCs. ( B ) GeNorm results between treated cell groups, i.e. hChondrocytes undergoing apoptosis, chondrogenic differentiated hBMSCs and hADSCs. ( C ) GeNorm results between all untreated and treated cell lines and types.

    Article Snippet: The PCR reactions were performed using a qRT-PCR LightCycler® 96 Instrument (Roche, Basel, Swiss), where the total volume per reaction was 10 μl, containing 10 μM of each reference primer (Table ), the corresponding diluted cDNA and 2x FastStart Essential DNA Green Master (Roche).

    Techniques: Expressing, Quantitative RT-PCR

    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hBMSCs and ( B ) chondrogenic differentiated hBMSCs separately or ( C ) both cell lines combined.

    Journal: Scientific Reports

    Article Title: Recommendations for improving accuracy of gene expression data in bone and cartilage tissue engineering

    doi: 10.1038/s41598-018-33242-z

    Figure Lengend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hBMSCs and ( B ) chondrogenic differentiated hBMSCs separately or ( C ) both cell lines combined.

    Article Snippet: The PCR reactions were performed using a qRT-PCR LightCycler® 96 Instrument (Roche, Basel, Swiss), where the total volume per reaction was 10 μl, containing 10 μM of each reference primer (Table ), the corresponding diluted cDNA and 2x FastStart Essential DNA Green Master (Roche).

    Techniques: Expressing, Quantitative RT-PCR

    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) untreated rat rectus abdominis muscle tissue, ( B ) rat rectus abdominis muscle tissue treated with osteogenic medium, and ( C ) both normal and treated rat muscle tissue.

    Journal: Scientific Reports

    Article Title: Recommendations for improving accuracy of gene expression data in bone and cartilage tissue engineering

    doi: 10.1038/s41598-018-33242-z

    Figure Lengend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) untreated rat rectus abdominis muscle tissue, ( B ) rat rectus abdominis muscle tissue treated with osteogenic medium, and ( C ) both normal and treated rat muscle tissue.

    Article Snippet: The PCR reactions were performed using a qRT-PCR LightCycler® 96 Instrument (Roche, Basel, Swiss), where the total volume per reaction was 10 μl, containing 10 μM of each reference primer (Table ), the corresponding diluted cDNA and 2x FastStart Essential DNA Green Master (Roche).

    Techniques: Expressing, Quantitative RT-PCR

    Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hADSCs and ( B ) chondrogenic differentiated hADSCs separately or ( C ) both untreated and treated hADSCs cell lines combined.

    Journal: Scientific Reports

    Article Title: Recommendations for improving accuracy of gene expression data in bone and cartilage tissue engineering

    doi: 10.1038/s41598-018-33242-z

    Figure Lengend Snippet: Average expression stability ( A1 , B1 , C1 ) and optimal number of reference genes for normalization ( A2 , B2 , C2 ) for qRT-PCR assays, utilizing the GeNorm algorithm, for ( A ) normal hADSCs and ( B ) chondrogenic differentiated hADSCs separately or ( C ) both untreated and treated hADSCs cell lines combined.

    Article Snippet: The PCR reactions were performed using a qRT-PCR LightCycler® 96 Instrument (Roche, Basel, Swiss), where the total volume per reaction was 10 μl, containing 10 μM of each reference primer (Table ), the corresponding diluted cDNA and 2x FastStart Essential DNA Green Master (Roche).

    Techniques: Expressing, Quantitative RT-PCR

    FOXO1 represses T-bet and miR-31 in Th1 cells. (A) Correlation between miR-31 expression normalized to snU6 and Foxo1 expression normalized to Hprt determined five times in 6 day intervals from naive to Th1 rep cells (qRT-PCR) ( n = 15 from one experiment, p value is depicted in the figure). (B) Foxo1 , Foxo3 and pri-miR-31 expression normalized to Hprt in repeatedly (two rounds of stimulation) activated Th1 cells, treated with a pool of 8 siRNAs specific for Foxo1 and Foxo3 or a SI-SCR control, analyzed 48 h after siRNA treatment by qRT-PCR, presented relative to the SI-SCR control. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, * p ≤ 0.05, ** p ≤ 0.01) (C) Klf2, Sell, Cd69 and pri-miR-31 expression normalized to Hprt in activated CD4 + cells transduced 36–40 h post activation with a retroviral vector expressing a constitutive active FOXO1 (FOXO1A3) or an empty control vector (RV). Cells were cultured under Th1 polarizing conditions for additional 48 h. Expression was analyzed by qRT-PCR 48 h post transduction and is presented relative to RV. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01) (D) Representative intracellular protein staining and T-Bet protein expression in the samples analyzed in (C) , presented as MFI of T-Bet, normalized to RV assessed by flow cytometry. Data is shown as mean +SEM, n = 5–11 pooled from three independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01). (E) Foxo1 expression normalized to Hprt in Th1 rep cells activated with αCD3/28 under Th1 polarizing conditions for 48 h ±TGFβ, presented relative to values obtained from untreated Th1 rep cells determined by qRT-PCR. Data is shown as mean +SEM, n = 9 pooled from 3 independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01, *** p ≤ 0.001). MiR-31 expression normalized to snU6 in the same cells, presented relative to Th1 rep cells before reactivation assessed by qRT-PCR. Data is shown as mean +SEM, n = 9 pooled from three independent experiments (One-way Anova with Dunn's test for multiple comparison, * p ≤ 0.05, *** p ≤ 0.001). (F) Representative intracellular protein staining of Th1 rep cells activated with αCD3/28 under Th1 polarizing conditions for 48 h ±TGFβ and T-Bet protein expression, presented as MFI of T-Bet, normalized to untreated Th1 rep cells. Data is shown as mean +SEM, n = 2–3 pooled from four independent experiments (Mann-Whitney test for unpaired data, *** p ≤ 0.001). Ifng expression normalized to Hprt in the samples analyzed in (A) , presented relative to RV. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, * p ≤ 0.05, ** p ≤ 0.01). (G) Representative intracellular FOXP3 protein staining of Th1 rep cells activated with αCD3/28 in Th1 polarizing conditions for 48 h ±TGF-β.

    Journal: Frontiers in Immunology

    Article Title: MicroRNA-31 Reduces the Motility of Proinflammatory T Helper 1 Lymphocytes

    doi: 10.3389/fimmu.2018.02813

    Figure Lengend Snippet: FOXO1 represses T-bet and miR-31 in Th1 cells. (A) Correlation between miR-31 expression normalized to snU6 and Foxo1 expression normalized to Hprt determined five times in 6 day intervals from naive to Th1 rep cells (qRT-PCR) ( n = 15 from one experiment, p value is depicted in the figure). (B) Foxo1 , Foxo3 and pri-miR-31 expression normalized to Hprt in repeatedly (two rounds of stimulation) activated Th1 cells, treated with a pool of 8 siRNAs specific for Foxo1 and Foxo3 or a SI-SCR control, analyzed 48 h after siRNA treatment by qRT-PCR, presented relative to the SI-SCR control. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, * p ≤ 0.05, ** p ≤ 0.01) (C) Klf2, Sell, Cd69 and pri-miR-31 expression normalized to Hprt in activated CD4 + cells transduced 36–40 h post activation with a retroviral vector expressing a constitutive active FOXO1 (FOXO1A3) or an empty control vector (RV). Cells were cultured under Th1 polarizing conditions for additional 48 h. Expression was analyzed by qRT-PCR 48 h post transduction and is presented relative to RV. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01) (D) Representative intracellular protein staining and T-Bet protein expression in the samples analyzed in (C) , presented as MFI of T-Bet, normalized to RV assessed by flow cytometry. Data is shown as mean +SEM, n = 5–11 pooled from three independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01). (E) Foxo1 expression normalized to Hprt in Th1 rep cells activated with αCD3/28 under Th1 polarizing conditions for 48 h ±TGFβ, presented relative to values obtained from untreated Th1 rep cells determined by qRT-PCR. Data is shown as mean +SEM, n = 9 pooled from 3 independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01, *** p ≤ 0.001). MiR-31 expression normalized to snU6 in the same cells, presented relative to Th1 rep cells before reactivation assessed by qRT-PCR. Data is shown as mean +SEM, n = 9 pooled from three independent experiments (One-way Anova with Dunn's test for multiple comparison, * p ≤ 0.05, *** p ≤ 0.001). (F) Representative intracellular protein staining of Th1 rep cells activated with αCD3/28 under Th1 polarizing conditions for 48 h ±TGFβ and T-Bet protein expression, presented as MFI of T-Bet, normalized to untreated Th1 rep cells. Data is shown as mean +SEM, n = 2–3 pooled from four independent experiments (Mann-Whitney test for unpaired data, *** p ≤ 0.001). Ifng expression normalized to Hprt in the samples analyzed in (A) , presented relative to RV. Data is shown as mean +SEM, n = 6 pooled from two independent experiments (Mann-Whitney test for unpaired data, * p ≤ 0.05, ** p ≤ 0.01). (G) Representative intracellular FOXP3 protein staining of Th1 rep cells activated with αCD3/28 in Th1 polarizing conditions for 48 h ±TGF-β.

    Article Snippet: Expression values of mRNAs were assessed by SYBR Green based qRT-PCR (Roche) using the following primer pairs: hypoxanthine guanine phosphoribosyltransferase (HPRT) forward 5′-TCCTCCTCAGACCGCTTTT-3′, HPRT reverse 5′-CATAACCTGGTTCATCATCGC-3′, Tbx21 forward 5′-TCCTGCAGTCTCTCCACAAGT-3′, Tbx21 reverse 5′-CAGCTGAGTGATCTCTGCGT-3′, FOXO1 forward 5′-CGGGCTGGAAGAATTCAATTC-3′, FOXO1 reverse, 5′-AGTTCCTTCATTCTGCACTCGAA-3′.

    Techniques: Expressing, Quantitative RT-PCR, MANN-WHITNEY, Activation Assay, Plasmid Preparation, Cell Culture, Transduction, Staining, Flow Cytometry, Cytometry

    Significant upregulation of miR-31 targets after knock-down of miR-31. (A) Schematic overview of the method to determine the fraction of putative miR-31 target mRNA molecules that contain at least one miR-31 bs in their 3′-UTR. Depicted is the coverage (black bars, middle row) of n exons and the 3′-UTR (upper row) containing the miR-31 bs (indicated in blue). The ratio is calculated from the median coverage of the exons and the coverage of the miR-31 bs in the 3′-UTR (bottom row). (B) 421 putative miR-31 targets were grouped according to the ratio determined in (A) . (C) MiR-31 expression in Th1 rep cells 24, 48, and 72 h after activation with αCD3/28 and treatment with Antagomir-31 or Antagomir-SCR normalized to snU6 determined by qRT-PCR. Data is shown as mean +SEM, n = 11, pooled from five independent experiments (Mann-Whitney test for unpaired data, *** p ≤ 0.001). (D) GSEA with the PT- and PT 50 - gene-sets and the transcriptome data of Th1 rep cells 36, 48, and 72 h after activation with αCD3/28 and treatment with Antagomir-31 or Antagomir-SCR. Data is shown as enrichment curves with each putative target gene (PT, black; PT 50 , green) in ranked order from most upregulated (left) to most downregulated (right). Nominal p -values are depicted in the figure. (E) Welch's test of nominal enrichment scores (NES) from 1,000 independent GSEA's each using a randomly chosen subset of PT with sizes equal to the size of the PT 50 set, p -value is depicted in the figure.

    Journal: Frontiers in Immunology

    Article Title: MicroRNA-31 Reduces the Motility of Proinflammatory T Helper 1 Lymphocytes

    doi: 10.3389/fimmu.2018.02813

    Figure Lengend Snippet: Significant upregulation of miR-31 targets after knock-down of miR-31. (A) Schematic overview of the method to determine the fraction of putative miR-31 target mRNA molecules that contain at least one miR-31 bs in their 3′-UTR. Depicted is the coverage (black bars, middle row) of n exons and the 3′-UTR (upper row) containing the miR-31 bs (indicated in blue). The ratio is calculated from the median coverage of the exons and the coverage of the miR-31 bs in the 3′-UTR (bottom row). (B) 421 putative miR-31 targets were grouped according to the ratio determined in (A) . (C) MiR-31 expression in Th1 rep cells 24, 48, and 72 h after activation with αCD3/28 and treatment with Antagomir-31 or Antagomir-SCR normalized to snU6 determined by qRT-PCR. Data is shown as mean +SEM, n = 11, pooled from five independent experiments (Mann-Whitney test for unpaired data, *** p ≤ 0.001). (D) GSEA with the PT- and PT 50 - gene-sets and the transcriptome data of Th1 rep cells 36, 48, and 72 h after activation with αCD3/28 and treatment with Antagomir-31 or Antagomir-SCR. Data is shown as enrichment curves with each putative target gene (PT, black; PT 50 , green) in ranked order from most upregulated (left) to most downregulated (right). Nominal p -values are depicted in the figure. (E) Welch's test of nominal enrichment scores (NES) from 1,000 independent GSEA's each using a randomly chosen subset of PT with sizes equal to the size of the PT 50 set, p -value is depicted in the figure.

    Article Snippet: Expression values of mRNAs were assessed by SYBR Green based qRT-PCR (Roche) using the following primer pairs: hypoxanthine guanine phosphoribosyltransferase (HPRT) forward 5′-TCCTCCTCAGACCGCTTTT-3′, HPRT reverse 5′-CATAACCTGGTTCATCATCGC-3′, Tbx21 forward 5′-TCCTGCAGTCTCTCCACAAGT-3′, Tbx21 reverse 5′-CAGCTGAGTGATCTCTGCGT-3′, FOXO1 forward 5′-CGGGCTGGAAGAATTCAATTC-3′, FOXO1 reverse, 5′-AGTTCCTTCATTCTGCACTCGAA-3′.

    Techniques: Expressing, Activation Assay, Quantitative RT-PCR, MANN-WHITNEY

    MiR-31 targets a set of genes involved in cytoskeletal rearrangement and miR-31 inhibition increases the motility of Th1 rep cells. (A) GSEA of the transcriptome data obtained from Th1 rep cells 72 h after activation with αCD3/28 and treatment with Antagomir-31 or Antagomir-SCR with the KEGG-pathway database (v. 6.0) used as source for gene-sets. Data of two significantly enriched gene-sets is shown as enrichment curves with all genes in ranked order from most upregulated (left) to most downregulated (right). Nominal p -values are depicted in the figure. (B) Network of validated functional interactions among positively correlated miR-31 targets (green rimmed; Figure 2D ) and the genes defining the gene set “regulation of actin cytoskeleton” (red). The resulting network was adapted to T cells (also see methods). Genes without interactions are not included. (C) QRT-PCR of target mRNA expression in reactivated Th1 rep cells 72 h after antagomir treatment relative to Hprt and normalized to Antagomir-SCR treated control. Data is shown as mean +SEM, n = 12 (for Lats2, RhoA, Stk40, Ywhae ) pooled from four independent experiments, or n = 6 (for Ablim1, Cd28, Cdc42, Eif4ebp2, LPP, Ppp2r2a, Ppp3ca, Rac1 ) pooled from two independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01, * p ≤ 0.05). (D,E) Transwell migration assays with an ICAM-1 coated membrane (10 μg/ml) and CXCL10 (100 ng/ml) in the lower compartment for once and repeatedly activated Th1 cells, 72 h after reactivation with αCD3/28 (D) and Th1 rep cells, 72 h after antagomir treatment and reactivation with αCD3/28 (E) , assessed by flow cytometry, normalized to inserted cell number. Data is shown as mean +SEM, n = 16–18 pooled from four independent experiments (Mann-Whitney test for unpaired data, *** p ≤ 0.001).

    Journal: Frontiers in Immunology

    Article Title: MicroRNA-31 Reduces the Motility of Proinflammatory T Helper 1 Lymphocytes

    doi: 10.3389/fimmu.2018.02813

    Figure Lengend Snippet: MiR-31 targets a set of genes involved in cytoskeletal rearrangement and miR-31 inhibition increases the motility of Th1 rep cells. (A) GSEA of the transcriptome data obtained from Th1 rep cells 72 h after activation with αCD3/28 and treatment with Antagomir-31 or Antagomir-SCR with the KEGG-pathway database (v. 6.0) used as source for gene-sets. Data of two significantly enriched gene-sets is shown as enrichment curves with all genes in ranked order from most upregulated (left) to most downregulated (right). Nominal p -values are depicted in the figure. (B) Network of validated functional interactions among positively correlated miR-31 targets (green rimmed; Figure 2D ) and the genes defining the gene set “regulation of actin cytoskeleton” (red). The resulting network was adapted to T cells (also see methods). Genes without interactions are not included. (C) QRT-PCR of target mRNA expression in reactivated Th1 rep cells 72 h after antagomir treatment relative to Hprt and normalized to Antagomir-SCR treated control. Data is shown as mean +SEM, n = 12 (for Lats2, RhoA, Stk40, Ywhae ) pooled from four independent experiments, or n = 6 (for Ablim1, Cd28, Cdc42, Eif4ebp2, LPP, Ppp2r2a, Ppp3ca, Rac1 ) pooled from two independent experiments (Mann-Whitney test for unpaired data, ** p ≤ 0.01, * p ≤ 0.05). (D,E) Transwell migration assays with an ICAM-1 coated membrane (10 μg/ml) and CXCL10 (100 ng/ml) in the lower compartment for once and repeatedly activated Th1 cells, 72 h after reactivation with αCD3/28 (D) and Th1 rep cells, 72 h after antagomir treatment and reactivation with αCD3/28 (E) , assessed by flow cytometry, normalized to inserted cell number. Data is shown as mean +SEM, n = 16–18 pooled from four independent experiments (Mann-Whitney test for unpaired data, *** p ≤ 0.001).

    Article Snippet: Expression values of mRNAs were assessed by SYBR Green based qRT-PCR (Roche) using the following primer pairs: hypoxanthine guanine phosphoribosyltransferase (HPRT) forward 5′-TCCTCCTCAGACCGCTTTT-3′, HPRT reverse 5′-CATAACCTGGTTCATCATCGC-3′, Tbx21 forward 5′-TCCTGCAGTCTCTCCACAAGT-3′, Tbx21 reverse 5′-CAGCTGAGTGATCTCTGCGT-3′, FOXO1 forward 5′-CGGGCTGGAAGAATTCAATTC-3′, FOXO1 reverse, 5′-AGTTCCTTCATTCTGCACTCGAA-3′.

    Techniques: Inhibition, Activation Assay, Functional Assay, Quantitative RT-PCR, Expressing, MANN-WHITNEY, Migration, Flow Cytometry, Cytometry

    TCR/CD28 induced expression of mir-31 is increased by T-Bet and IFN-γ. (A) MiR-31 expression kinetics normalized to snU6 after the first activation of naive CD4 + (left panel) or reactivation of Th1 rep cells (right panel) with αCD3/28, presented relative to values obtained from naive CD4 + cells ex vivo , determined by qRT-PCR. Data is shown as mean ±SEM, n = 8–12 pooled from 3 to 4 independent experiments (One-way Anova with Mann-Whitney test for unpaired data, * p ≤ 0.05, ** p ≤ 0.01). (B) RNA-Seq coverage of the miR-31 gene locus in Th1 rep cells (upper row) and analysis of published ChIP-seq data from Th1 cells for p300 ( 28 ), H3K4me3 and H3K27me3 ( 29 ) using the Cistrome Browser (lower row). (C) Analysis of the putative promoter region as determined in (B) using published ChIP-Seq data obtained from Th1 cells for T-Bet ( 34 ), STAT1 ( 28 ), and STAT4 ( 33 ) and from naive CD4 + T cells ( 35 ), as well as predicted conserved binding sites for these transcription factors obtained from ECR Browser. (D) Schematic overview of the murine miR-31 gene locus and the resulting primary transcript as analyzed in (B) . (E) MiR-31 expression normalized to Hprt in naive CD4 + cells activated with αCD3/28 in Th1 polarizing conditions for 48 h ± IFN-γ (10 ng/ml), presented relative to values obtained from naive CD4 + cells ex vivo determined by qRT-PCR. Data is shown as mean ±SEM, n = 4–8 pooled from two independent experiments (One-way Anova with Dunn's test for multiple comparison, ** p ≤ 0.01).

    Journal: Frontiers in Immunology

    Article Title: MicroRNA-31 Reduces the Motility of Proinflammatory T Helper 1 Lymphocytes

    doi: 10.3389/fimmu.2018.02813

    Figure Lengend Snippet: TCR/CD28 induced expression of mir-31 is increased by T-Bet and IFN-γ. (A) MiR-31 expression kinetics normalized to snU6 after the first activation of naive CD4 + (left panel) or reactivation of Th1 rep cells (right panel) with αCD3/28, presented relative to values obtained from naive CD4 + cells ex vivo , determined by qRT-PCR. Data is shown as mean ±SEM, n = 8–12 pooled from 3 to 4 independent experiments (One-way Anova with Mann-Whitney test for unpaired data, * p ≤ 0.05, ** p ≤ 0.01). (B) RNA-Seq coverage of the miR-31 gene locus in Th1 rep cells (upper row) and analysis of published ChIP-seq data from Th1 cells for p300 ( 28 ), H3K4me3 and H3K27me3 ( 29 ) using the Cistrome Browser (lower row). (C) Analysis of the putative promoter region as determined in (B) using published ChIP-Seq data obtained from Th1 cells for T-Bet ( 34 ), STAT1 ( 28 ), and STAT4 ( 33 ) and from naive CD4 + T cells ( 35 ), as well as predicted conserved binding sites for these transcription factors obtained from ECR Browser. (D) Schematic overview of the murine miR-31 gene locus and the resulting primary transcript as analyzed in (B) . (E) MiR-31 expression normalized to Hprt in naive CD4 + cells activated with αCD3/28 in Th1 polarizing conditions for 48 h ± IFN-γ (10 ng/ml), presented relative to values obtained from naive CD4 + cells ex vivo determined by qRT-PCR. Data is shown as mean ±SEM, n = 4–8 pooled from two independent experiments (One-way Anova with Dunn's test for multiple comparison, ** p ≤ 0.01).

    Article Snippet: Expression values of mRNAs were assessed by SYBR Green based qRT-PCR (Roche) using the following primer pairs: hypoxanthine guanine phosphoribosyltransferase (HPRT) forward 5′-TCCTCCTCAGACCGCTTTT-3′, HPRT reverse 5′-CATAACCTGGTTCATCATCGC-3′, Tbx21 forward 5′-TCCTGCAGTCTCTCCACAAGT-3′, Tbx21 reverse 5′-CAGCTGAGTGATCTCTGCGT-3′, FOXO1 forward 5′-CGGGCTGGAAGAATTCAATTC-3′, FOXO1 reverse, 5′-AGTTCCTTCATTCTGCACTCGAA-3′.

    Techniques: Expressing, Activation Assay, Ex Vivo, Quantitative RT-PCR, MANN-WHITNEY, RNA Sequencing Assay, Chromatin Immunoprecipitation, Binding Assay

    MiR-31 is upregulated in murine Th1 rep cells, and in memory Th cells from the synovial fluid of RA patients. (A) MiR-31 expression in once (day 6) and repeatedly (three rounds of restimulation with 6 day intervals) activated Th1, Th2, Th17, and ex vivo isolated naive CD4 + cells normalized to snU6 determined by qRT-PCR. Each data point represents an independent experiment ( n = 12 [naive and Th1], 5 [Th2], 4 [Th17]) (Wilcoxon-Test for paired data, *** p ≤ 0.001). (B) MiR-31 expression normalized to snU6 in CD3 + CD4 + CD14 − CD45RO + T cells isolated from the synovial fluid of patients suffering from RA or blood from healthy control (HC) donors ex vivo or after 3 h of restimulation with PMA/ionomycin (P/I) ( n = 5 RA; n = 4 HC) determined by qRT-PCR. Each data point represents an individual donor, horizontal bar: median (Mann-Whitney test for unpaired data, * p ≤ 0.05).

    Journal: Frontiers in Immunology

    Article Title: MicroRNA-31 Reduces the Motility of Proinflammatory T Helper 1 Lymphocytes

    doi: 10.3389/fimmu.2018.02813

    Figure Lengend Snippet: MiR-31 is upregulated in murine Th1 rep cells, and in memory Th cells from the synovial fluid of RA patients. (A) MiR-31 expression in once (day 6) and repeatedly (three rounds of restimulation with 6 day intervals) activated Th1, Th2, Th17, and ex vivo isolated naive CD4 + cells normalized to snU6 determined by qRT-PCR. Each data point represents an independent experiment ( n = 12 [naive and Th1], 5 [Th2], 4 [Th17]) (Wilcoxon-Test for paired data, *** p ≤ 0.001). (B) MiR-31 expression normalized to snU6 in CD3 + CD4 + CD14 − CD45RO + T cells isolated from the synovial fluid of patients suffering from RA or blood from healthy control (HC) donors ex vivo or after 3 h of restimulation with PMA/ionomycin (P/I) ( n = 5 RA; n = 4 HC) determined by qRT-PCR. Each data point represents an individual donor, horizontal bar: median (Mann-Whitney test for unpaired data, * p ≤ 0.05).

    Article Snippet: Expression values of mRNAs were assessed by SYBR Green based qRT-PCR (Roche) using the following primer pairs: hypoxanthine guanine phosphoribosyltransferase (HPRT) forward 5′-TCCTCCTCAGACCGCTTTT-3′, HPRT reverse 5′-CATAACCTGGTTCATCATCGC-3′, Tbx21 forward 5′-TCCTGCAGTCTCTCCACAAGT-3′, Tbx21 reverse 5′-CAGCTGAGTGATCTCTGCGT-3′, FOXO1 forward 5′-CGGGCTGGAAGAATTCAATTC-3′, FOXO1 reverse, 5′-AGTTCCTTCATTCTGCACTCGAA-3′.

    Techniques: Expressing, Ex Vivo, Isolation, Quantitative RT-PCR, MANN-WHITNEY