Tp800 qpcr system

Total RNAs were extracted from fresh frozed tissue using Trizol reagent (Invitrogen, USA) and reverse transcribed using ReverTra Ace qPCR RT Kit (TOYOBO, Japan), according to the manufacturers’ instructions. The resulting complementary DNA (cDNA) was used for real-time PCR using the SYBR Premix Ex Taq II (Applied TaKaRa, Japan) in triplicates. PCR and data collection were performed on the TP800 qPCR System (Takara, Japan). All data were normalized to an endogenous control (GAPDH). The relative value for the target gene compared to its calibrator is expressed as 2^-(Ct-Cc) (Ct and Cc are the mean threshold cycle differences after normalizing to GAPDH)). Primers used in PCR are showed in Table 1.

Lentiviral transduction efficiency was validated using qPCR analysis after transduction for 72 h. Total RNA was extracted using TRIzol reagent and reverse transcribed using M-MLV Reverse Transcriptase according to the manufacturer’s instructions. The resulting complementary (c)DNA was used for qPCR analysis using SYBR-Green PCR Master mix. qPCR analysis was performed in triplicate using the TP800 qPCR System (Takara Bio Inc.). Target gene expression was normalized to that of the endogenous control GAPDH. The relative quantitative expression of the target gene compared with GAPDH was expressed as 2^−(Ct-Cc) (Ct and Cc represent the mean threshold cycle differences following normalization to GAPDH). The qPCR primer sequences were as follows: Forward: 5′-TTCAGAAGGTGGCTCAATGC-3′ and reverse: 5′-GGAGTGTTGGAGAAGTCATATTAC-3′ for KLF8; and forward: 5′-TGACTTCAACAGCGACACCCA-3′ and reverse: 5′-GGAGTGTTGGAGAAGTCATATTAC-3′ for GAPDH.