High capacity cdna reverse transcription protocol

For quantitative analysis of gene expression, total RNA was extracted using RNeasy protocol (Qiagen) from cultured or treated cells. RNA was treated with DNase (Promega), and the concentration was measured by Nanodrop (Thermo). 1 μg total RNA was used to generate complementary DNA using the high-capacity cDNA reverse transcription protocol (Takara). Quantitative real-time PCR was then performed using reaction mixtures of cDNA, primers, and SYBR Green reagent (Takara) with the ABI StepOne system (PerkinElmer). PCR was done in triplicate, and SDs representing experimental errors were calculated. All data were analyzed using ABI PRISM SDS 2.0 software (PerkinElmer). The sequence of primers are as follows: SAE1-F: 5′-CAGTATGACCGACAGATCCGC-3′, R: 5′-GGCAACCTGAGCCTTTGATCT-3′; SAE2-F: 5′-CCACATCGACCTGATTGATCTG-3′, R: 5′-GGCAACCTGAGCCTTTGATCT-3′; UBC9-F: 5′-GGAGGAAGGACCACCCTTTTG-3′, R: 5′-GGATAGCGCACTCCCAGTT-3′; SUMO1-F: 5′-ATTGGACAGGATAGCAGTGAGA-3′, R: 5′-TCCCAGTTCTTTCGGAGTATGA-3′; SUMO2-F: 5′-AAGGAAGGAGTCAAGACTGAGAA-3′, R: 5′-CGGAATCTGATCTGCCTCATTG-3′; SENP2-F: 5′-CACCAAATGGAGCCTGATCT-3′, R: 5′-CTTCCGTTCTTCTGTCCTTCTC-3′.