Quantifying Gene Expression Using RT-qPCR

Using the RNA isolation kit (Takara Biomedical Technology [Beijing] Co., Ltd.) to extract total RNA from cell lines, we used MMLV reverse transcriptase to synthesize cDNA using the SYBR Green reaction system (Takara Biomedical Technology [Beijing] Co., Ltd.) to amplify cDNA 37 (link). GAPDH was used as an internal reference to analyze the differences in mRNA expression levels of RB1, CDKN2B, CDKN2AIP, PCNA, KIAA0101, and MCM7. The PCR sequences were as follows: RB1: sense: 5'-GGAAGCAACCCTCCTAAACC-3', antisense: 5'-TTTCTGCTTTTGCATTCGTG-3'; CDKN2B: sense: 5'-CCAGAAGCAATCCAGGCGCG-3', antisense: 5'-CGTTGGCAGCClTCATCG-3'; CDKN2AIP sense: 5′- ATGGGCCAACAACGTGTTTC-3′, antisense: 5′-GAAAACAGGGGCATCCTCCA-3′; PCNA: sense: 5′-CCATCCTCAAGAAGGTGTTGG-3′, antisense: 5′-GTGTCCCATATCCGCAATTTTAT-3′; KIAA0101: sense: 5′-CCCAGGCAACATAGCGTAAA-3′, antisense: 5′-GGATGGTCTCGATCTCCTGA-3′; MCM7: sense: 5′-TCAATTTGTGAGAATGCCAGGCGC-3′, antisense: 5′-CACAGTTACCAACTTCCCCACAGA-3′; GAPDH: sense: 5′-TCCTGCACCACCAACTGCTTAG-3′, antisense: 5'-TCTTACTCCTTGGAGGCCATGT-3'.