Sybr green pcr mixture

Total RNA was prepared from cells using the RNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer's recommendation. The RNA was treated with DNaseI to remove residual genomic DNA and transcribed into cDNA using random primers and RevertAid H MinusM-MuLV reverse transcriptase (Fermentas, St. Leon-Rot, Germany). Real-time PCR was performed in duplicates with a SYBR Green PCR mixture (Qiagen/Promega, Mannheim, Germany). The cDNA was denatured for 15 min at 95 °C followed by 40 cycles of 95 °C for 15 s and 50 °C for 1 min using the 7000 ABI sequence detection system and gene specific primers. The signals were normalised to the signals for the housekeeping gene RibPO (34B4). See Supplementary Table 3 for primer sequences.

The siRNA transfections were performed according to the manufacturer’s instructions. As one of the human ACC cell lines, SW13 cells were transfected with 50 nmol of CENPF siRNA (siCENPF), CDK1 siRNA (siCDK1), or negative control siRNA (siNC) in a special medium (CM0451, Procell, China) for 48 h. Then, SW13 cells were lysed by TRIzol reagent (Invitrogen, USA) for total RNA isolation. The cDNA was obtained by reverse transcriptase kit (Invitrogen, USA). SYBR Green PCR Mixture (Qiagen, Germany) and specific primers were performed in ABI Prism 7500 analyzer (Applied Biosystems, USA). GAPDH was an endogenous reference gene. Three replicates were set for all reactions. The 2^−ΔΔCt method was applied to calculate the relative expression of CENPF or CDK1 in ACC samples. The details of primers were listed in Additional file 4: Table S4.