Mmtv reverse transcriptase

Total RNA was isolated from cells treated with each drug or siRNA using Sepasol-RNAI (Nacalai Tesque) according to the manufacturer’s instructions. Total RNA (2 μg) was reversely transcribed to complementary DNA (cDNA) in a 20 μl reaction volume with MMTV-reverse transcriptase (Promega) and oligo (dT) primers (Toyobo, Osaka, Japan). An equivalent volume of cDNA solution was used for quantitative RT–PCR. cDNA was amplified using an ABI 7300 real-time PCR system (Applied Biosystems, Foster City, CA, USA) with TaqMan Probes to CCND1 (Hs00765553_m1) and β2MG (Hs00984230_m1) (Applied Biosystems). The expression of cyclin D1 mRNA was normalized to that of β2MG mRNA in the same sample. All experiments shown were replicated three times.

RNA isolation and quantitative PCR were performed as previously described [42 (link)]. Briefly, total RNA was isolated from cells treated with the agent or siRNAs using Sepasol-RNA I (Nacalai Tesque) according to the manufacturer’s instructions. Total RNA (2 μg) was reverse-transcribed to complementary DNA (cDNA) in a 20 μL reaction volume with MMTV-reverse transcriptase (Promega) and oligo (dT) primers (Toyobo, Osaka, Japan). An equivalent volume of cDNA solution was used for quantitative PCR. cDNA was amplified using an ABI 7300 real-time PCR system (Applied Biosystems, Foster City, CA, USA) with TaqMan Probes for TYMS (Hs00426591_m1) and GAPDH (Hs02758991_g1) (Applied Biosystems). The expression of thymidylate synthase mRNA was normalized to that of GAPDH mRNA in the same sample.