Quantitative RT-PCR for Gene Expression Analysis

QRT-PCR was performed as previously described [22 (link), 23 (link)]. Briefly, total RNAs were isolated and purified with TRIzol reagent (Invitrogen, USA). Complementary DNA was synthesized from the total RNA using HiScript II Q RT Super Mix kit (Vazyme, Nanjing, China). QPCR was carried out with Step One Plus Real-Time PCR System (Applied Biosystems) using ChamQ Universal SYBR qPCR Master Mix kit (Vazyme, Nanjing, China). PCR conditions were 95°C for 30 s, followed by 40 cycles at 95°C for 10 s and 60°C for 30 s. PCR primers used were: p27, 5’-TCTCAGGCAAACTCTGAGGA-3’ (F) and 5’-CTTCCTCATCCCTGGACACT-3’ (R) [24 (link)]; β-actin, 5’-ACATCCGTAAAGACCTCTATGCCAACA-3’ (F) and 5’-GTGCTAGGAGCCAGGGCAGTAATCT-3’ (R). The mRNA expression level was normalized to the expression level of the housekeeping gene β-actin. All analyses were performed by using the 2^−ΔΔCt method [25 (link)].