Maxima sybr green rox qpcr master mix

Total RNA was isolated from PCa cells (LNCaP, LNCaP/Docetaxel or primary PCa cells) with or without miR-122 inhibitor or mimic, and/or combined with PKM2 overexpression (oePKM2) lentivirus using TRIzol^® reagent (cat. no. 15996026; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Following RNA quanrification and integrity confirmation, ~1 µg of RNA was reversed transcribed into cDNA using the RevertAid First Strand cDNA Synthesis kit (cat. no. K1622; Thermo Fisher Scientific, Inc.) using the following heat cycle: 37˚C for 5 min; 55˚C for 15 min and 85˚C for 5 min. qPCR was performed on a 7300 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) with a miRNA RT-PCR Detection Kit (cat. no. AOMD-Q020; GeneCopoeia, Inc.) or a Maxima SYBR Green/ROX qPCR master mix (cat. no. K0223; Thermo Fisher Scientific, Inc.). The following thermocycling conditions were used for the qPCR: Initial denaturation at 95˚C for 10 min, 40 cycles of 95˚C for 15 sec and 60˚C for 45 sec and a final extension step at 95˚C for 15 sec. miR-122 expression were normalized to U6 levels and PKM2 expression were normalized to GAPDH levels using the 2^-ΔΔCq method (41 (link)). The primer pairs used for the qPCR are listed in Table I.