Quantitative RT-PCR for Gene Expression

Total RNA was extracted from the HeLa and SiHa cells using TRIzol (Invitrogen; Milan, Italy) according to manufacturer’s protocol, and 500 ng was used to obtain cDNA through reverse transcription using PrimeScript RTMasterMix (Promega; Madison, WI, United States). Quantitative RT-PCR was conducted using an SYBR Green PCR master mix (Roche; Basel, Switzerland) on the CFX96 Real-Time PCR Detection System (Bio-Rad; Hercules, CA, United States). PCR amplification was performed with the specific primer sets as described previously [12 (link)]. Relative expression was normalized to the expression of β-actin. The 2^−ΔΔCt method was used for relative quantification of gene expression. The primers used in the studies were: PKM2 sense, 5′-ATGTCGAAGCCCCATAGTGAA-3′, PKM2 antisense, 5′-TGGGTGGTGAATCAATGTCCA-3′, β-actin sense, 5′-CTCCATCCTGGCCTCGCTGT-3′, β-actin antisense, 5′-GCTGTCACCTTCACCGTTCC-3′.