Fast sybr green rt pcr kit

For quantitative real-time-PCR assays, total RNA was purified using SV Total RNA Isolation (Promega, Madison, WI, USA) and its integrity was assessed by capillary electrophoresis (Agilent, Santa Clara, CA, USA). RNA (400 ng) was converted to cDNA using random primers and Superscript III (Invitrogen, Carlsbad, CA, USA). Amplification was carried out in triplicates with a 7900HT Fast Real Time PCR System (Applied Biosystems) using the Fast SYBR Green RT-PCR kit (Applied Biosystems) and a standard 2-step protocol. The primers specific for each gene were designed and analysed with Primer3 (freeware) and Vector NTI (Invitrogen, freeware). Identity of the amplicons was confirmed by their dissociation profiles and gel analysis. Quantitative PCR standard curves were constructed by using serial dilutions of muscle cDNAs, using at least four dilution points and the efficiency of all primer sets was between 1.8 and 2.2. The data were normalized against Rplp0 housekeeping gene. Primers are listed in Supplementary Table 2.

After 7 days of culture total RNA was extracted from PACs using RNeasy kit (Qiagen), following the manufacturer’s protocol. RNA quantity was determined on a Nanodrop Spectrometer (termo Fisher scientific Inc) (using 1 OD260 = 40 μg RNA). A260/A280 ratios were also calculated for each sample. RNA was reverse transcribed to generate cDNA using the First-Strand cDNA Synthesis Kit from Invitrogen following the manufacturer’s protocol. Samples were mixed by vortexing and briefly centrifuged and denaturated by incubation for 5 minutes at 70°C to prevent secondary structures of RNA. Samples were incubated on ice for 2 minutes to allow the primers to align. Gene-specific primer pairs were designed using Primer-BLAST (NCBI) and were each validated prior to use by gradient PCR and gel analysis to test for optimal annealing temperature, reaction efficiency and specificity (Table 
1). Duplicates of sample cDNA were then amplified on the 7900HT Fast Real-Time PCR System (Applied Biosystems) using the Fast SYBR Green RT-PCR kit (Applied Biosystems) in 96-wells plates (micro amp optical, Applied Biosystems). Expression data were normalized to the mean of housekeeping gene to control the variability in expression levels and were analyzed using the 2^-ΔCT method.