Total RNA was extracted from isolated hepatocyte as described above. To remove genomic DNA contaminants, 5μg total RNA was treated with 2.5 units DNAse (Roche Applied Science, Mannheim, German) for 15 minutes at 20°C followed by inactivation of DNAse enzyme at 70°C for 8 minutes. Then, cDNA was synthesized from total RNA using SuperScript® III First-Strand Synthesis System (Invitrogen) according to the manufacturer’s protocol. 20ng cDNA was loaded into each well in RT2 Profiler 96-well PCR array plates (PARN-006, QIAGEN, Valencia CA) and amplified using the ABI 7500 real-time PCR System. The PCR reaction was programmed as follows: initial denaturing at 95°C for 10 min followed by 95°C for 15 sec, 60°C for 1 min, cycled 40 times. The median cycle threshold value (Ct) was uploaded onto the SABioscience website (http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php ) and the fold change of each gene expression was calculated using the provided software according to manufacturer’s instruction. Additionally, subsets of genes with the values of fold change larger than 1.5 across the 3 comparisons (normal vs compensated cirrhotic hepatocytes, normal vs decompensated cirrhotic hepatocytes, compensated vs decompensated cirrhotic hepatocytes) were used to find possible signal pathways using Ingenuity Pathway Analysis software.
Rt2 profiler 96 well pcr array plates
Manufactured by Qiagen
Sourced in Spain, Germany
The RT2 Profiler 96-well PCR array plates are a tool used in real-time PCR (polymerase chain reaction) experiments. The plates contain pre-dispensed primer assays for the analysis of gene expression across multiple samples. The core function of these plates is to facilitate the simultaneous measurement and comparison of the expression levels of a panel of genes related to a specific biological process or disease pathway.
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3 protocols using rt2 profiler 96 well pcr array plates
1
Transcriptome Profiling of Cirrhotic Hepatocytes
2
PCR Array Analysis of cDNA Samples
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20 ng cDNA was loaded into each well in RT2 Profiler 96-well PCR array plates (PARN-008Z, QIAGEN, Valencia CA). The median cycle threshold value (CT) was uploaded onto the SABioscience website (http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php ) and the fold change of each gene expression was calculated using the provided software according to manufacturer's instruction.
3
Characterizing Th17 Immune Responses
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The different aspects of the study included different numbers of patients as illustrated (Figure 1 ). From the 40 patients, plasma and cell samples were processed in the laboratory within 4 h of phlebotomy. Plasma from a total of 31 out of 40 patients was analysed using the Th1/Th2/Th17 cytometric bead array (CBA) kit in an initial exploratory study. Based on this analysis, plasma samples from 23 patients were randomly selected for analysis of selected Th17 related cytokines including IL-6 using the MILLIPLEX® MAP Human Th17 Magnetic Bead Panel kit (Millipore™, Massachusetts, United States).
RNA was extracted from peripheral blood mononuclear cells (PBMCs) using the TriReagent® (Sigma Aldrich, Missouri, United States) method from 13 patients for screening of genes with the human innate and adaptive RT2 Profiler 96-well PCR array plates (QIAGEN, Hilden, Germany). Findings showed dose-dependent expression of the CCR8 gene with disease severity, prompting further analysis in 29 patients using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) to verify its roles. To characterize cell types into monocytes, lymphocytes, and granulocytes, seven patients were included in an antibody specific multicolour immunophenotyping flow cytometry experiment.
RNA was extracted from peripheral blood mononuclear cells (PBMCs) using the TriReagent® (Sigma Aldrich, Missouri, United States) method from 13 patients for screening of genes with the human innate and adaptive RT2 Profiler 96-well PCR array plates (QIAGEN, Hilden, Germany). Findings showed dose-dependent expression of the CCR8 gene with disease severity, prompting further analysis in 29 patients using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) to verify its roles. To characterize cell types into monocytes, lymphocytes, and granulocytes, seven patients were included in an antibody specific multicolour immunophenotyping flow cytometry experiment.
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