PMC:4502368 / 10910-12559 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/4502368","sourcedb":"PMC","sourceid":"4502368","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4502368","text":"Direction and magnitude of expression of three genes (SERPINH1, FOS, and CXCL8) in the pooled samples were confirmed with qPCR. RNA was treated with Deoxyribonuclease I, Amplification Grade (Life Technologies), to remove possible genomic DNA contamination according to manufacturer’s instructions. DNase-treated RNA was converted to cDNA using the SuperScript VILO cDNA Synthesis Kit (Life Technologies) according to the manufacturer’s instructions. Quantitative PCR was then performed using BioRad Chromo4 real-time detector in an 8 μl reaction [1 μM forward primer, 1 μM reverse primer, 1X Quantitect SYBR Green (Qiagen), and 5 ng cDNA]. Cycling parameters were 95° (15 min), 94° (15 sec), 58° (30 sec), 72° (30 sec) (data acquisition) × 45 cycles, followed by a melt curve between 55° and 95° to ensure a single amplicon was present (Table 1). Standard curves were performed for each primer pair to ensure PCR efficiency was between 95% and 105%. Three replicates were performed for each sample and two no-template controls were included on each plate, and a no-RT control was included for each sample. Quantitative PCR was also performed for selected genes on individual RNA samples to assess expression variation within a pooled treatment group (Supporting Information, Figure S1). Quantitative PCR results were normalized to the housekeeping gene EF1A after ensuring its expression level remained stable across samples and experimental conditions. Analysis of qPCR data was performed using the delta delta Ct method, and results for all three technical replicates for a given sample were required to be within 10% of each other to be included.","tracks":[]}