Qrt pcr assay

Based on manufacturer’s instructions, we extracted total RNA from cells using the TRIZOL reagent (Invitrogen, Carlsbad, CA, USA). The Reverse Transcription Kit was used to reverse-transcribe one microgram of total RNA into cDNA for use in the qRT-PCR assay (Takara, Dalian, China). With the use of the Fast Real-time PCR 7500 System(Applied Biosystems, Foster City, CA, USA), we were able to determine gene expression. After two minutes at 50°C, the PCR reaction was subjected to 40 cycles of 95°C for 15 seconds, followed by one minute at 60°C. The GAPDH gene was amplified to serve as an internal control. The relative quantification values for CKS2 were calculated by the 2^-ΔΔCt method. The primers were as follows: CKS2 sense: 5’-TTCGACGAACACTACGAGTACC-3’; CKS2 antisense: 5’- GGACACCAAGTCTCCTCCAC-3’; GAPDH sense: 5’-AGAAGGCT-GGGGCTCATTTG-3’; GAPDH antisense: 5’-AGGGGCCATCCACAGTCTTC-3’.

Total RNA was extracted and reversely transcribed into cDNA using the Reverse Transcription System Kit (TaKaRa Bio Inc., Otsu, Japan). The levels of RNA expression were quantified by qRT-PCR assay (TaKaRa) and calculated by the 2^−ΔΔCt method. The values were normalized to an endogenous control GAPDH.