Ecodry premix double primed kit

RNA was prepared from cells actively growing in 60 mm tissue culture plates (12556001; Fisher Scientific, Hanover Park, IL, USA). Total RNA was extracted using a combination of TRIzol reagent (15596026; Life Technologies, Grand Island, NY, USA) and an RNeasy mini kit (74104; Qiagen, Maryland, CA, USA) column for purification. The cDNA synthesis was performed from 1 μg of total RNA using the RNA to cDNA EcoDry Premix (Double Primed) kit (639549; Clontech, Mountain View, CA, USA). A 10-fold dilutions of cDNA was used as the template in qPCR assays, performed in triplicate on a Mastercycler Realplex (Eppendorf; Enfield, CT, USA) using the TB-Green Advantage qPCR Premix (639676; Clontech). The amplification conditions used were as follows: 95 °C for 2 min; 40 cycles of amplification (95 °C for 15 s, 60 °C for 40 s); and dissociation curve step (95 °C for 15 s, 60 °C for 15 s, 95 °C for 15 s). The linear regression line for nanograms of DNA was assessed from the relative fluorescent units (RFUs) at a threshold fluorescence value (Ct). Expression levels of target genes were quantified by comparing the RFU at the Ct to the standard curve and normalized by simultaneous amplification of 60S ribosomal protein L13α (Rpl13a), used as a housekeeping gene. The list of primers is provided in Table 1.