Sybr green master rox reagent

Total RNA was isolated from cells using the RNAprep cell kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s instructions. The purity and concentration of RNA were examined by a nucleic acid protein detector (Thermo Scientific, Waltham, USA). RNA samples were reverse transcribed using a RevertAid First Strand kit (Thermo Scientific). qRT-PCR was performed with a 7500 Fast Real-Time PCR System using SYBR Green Master (ROX) reagent (Roche, Basel, Switzerland). The sequences of the primers were as follows: CENPM forward 5′-CAGTCTCCAGAACACAGAGGAGTC-3′ and reverse 5′-CACCTGTGGCGAGGAAAC-3′; GAPDH forward 5′-ACAGCCTCAAGATCATCAGC-3′ and reverse 5′-GGTCATGAGTCCTTCCACGAT-3′. The data were analyzed using the 2
^−ΔΔCT method. GAPDH expression was used for the normalization of CENPM expression level.

Total RNA from cells was isolated according to the RNAprep cell kit instructions (Tiangen Biotech Co, Ltd.). The purity and concentration of RNA were examined by a nucleic acid protein detector (DeNovix, Inc.). RNA samples were reverse transcribed by a RevertAid First Strand kit (Thermo Fisher Scientific, Inc.). qRT-PCR was performed using a 7500 Fast Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) with SYBR Green Master (ROX) reagent (Roche Diagnostics). The thermocycling conditions were as follows: Initial denaturation at 95°C for 10 min; 40 of cycles of denaturation for 15 sec at 95°C, annealing for 1 min at 60°C and elongation for 1 min at 72°C; and final extension for 10 min at 72°C. GAPDH was used for the normalization of CENPM expression levels. The sequences of the GAPDH primers used in the present study were forward 5′-GGACCTGACCTGCCGTCTAG-3′ and reverse 5′-GTAGCCCAGGATGCCCTTGA-3′. The sequences of the CENPM primers used in the present study were forward 5′-CTGGCGGACTCGATGCTCAAAG-3′ and reverse 5′-CGATTCACACTGGAGGGCAAAGG-3′. The results are presented with CT values and were analyzed by the ΔΔCq method (19 (link)). Each sample was performed in triplicate.