Fast universal sybr green master rox

Total RNA from individuals was extracted with TRIzol reagent (Invitrogen, Thermo Fisher Scientific) as described by the manufacturer's instruction. One microgram of RNA was reverse transcribed into cDNA using oligo-dT and MMLV-RT (Moloney Murine Leukemia Virus Reverse Transcriptase, Invitrogen, Thermo Fisher Scientific). Before cDNA synthesis, genomic DNA in the samples was digested with DNAsa (Ambion, Thermo Fisher Scientific) according to the manufacturer's instructions. The cDNA was used as template for real-time PCR analysis. Reactions were performed on a Step-one Real-time PCR machine from Applied Biosystems (California, USA) with Fast Universal SYBR Green Master Rox (Roche, Merck). The primer sequences are listed in S2 Table . To verify the purity of the PCR products, a melting curve was produced after each run. LinRegPCR was the program employed for the analysis of real time RT-PCR data (21) . The transcript relative quantification results were determined from the ratio between the starting concentration value of the analysed mRNAs and the reference Actin mRNA in each sample. The mean and standard error was calculated from values of the transcript quantification obtained in each biological replicate.

Total RNA was extracted using Trizol isolation reagent (Invitrogen), and treated with RQ1 RNasefree DNase (Promega). One μg of total RNA was used for first-strand cDNA synthesis with an oligo(dT) primer and M-MLV reverse transcriptase (Promega). The primer sequences are listed in Supplementary Table S1. For quantitative RT-PCR, reactions were performed on a Step-one Realtime PCR machine from Applied Biosystems (California, USA) with Fast Universal SYBR Green Master Rox (Roche) to monitor double-stranded DNA synthesis. Relative transcript levels were determined for each sample and normalized against actin transcript levels.