Sybra green pcr master mix

RNA in cells was extracted by Trizol Reagent (Thermo Fisher Scienti c, USA). The extracted RNA was reversely transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scienti c, USA). According to instructions of the kit, qPCR analysis was done with SYBRA Green PCR Master Mix (Takara, Japan) in QuantStudio 3 (Thermo Fisher Scienti c, USA) qPCR instrument (primers: see Table 1). GAPDH was utilized as an internal reference gene for CDK1 expression, and U6 for miR-195-5p. RIPA lysis buffer was used to treat cells and isolate intracellular proteins. SDS-polyacrylamide gel electrophoresis was performed on the same amount of protein samples. Then, the proteins were blotted onto the PVDF membrane and sealed with 5% skim milk at room temperature for 1 h. Finally, membrane was co-incubated with primary antibody at 4 ℃ overnight. Antibodies were purchased from Abcam (UK): anti-CDK1 antibody (ab32094), anti-RPA32 antibody (ab109084), anti-p-RPA32 antibody (ab109394), and GAPDH antibody (ab181602). After the primary antibody was washed, membrane was cultured with the secondary antibody IgG H&L (HRP, ab6721) for 1 h at room temperature. Imaging was developed and photographed on ChemiDoc™ Touch Imaging System (Bio-RAD US) using a hypersensitive ECL kit, and nally analyzed using ImageJ.