RNA extraction and real-time PCR
RNA was extracted from the tissues of siRNA-treated mice with TRIzol (Invitrogen). We synthesized cDNA from 500 ng of total RNA using the PrimeScript™ RT reagent kit (TaKaRa, Otsu, Japan) per the manufacturer's protocol. Casz1 mRNA was analyzed by real-time PCR. One-tenth of the cDNA reaction was added to a final volume of 20 µL for each reaction containing SYBR Green I (TaKaRa). The primers for Casz1 and glyceraldehye-3-phosphate dehydrogenase (GAPDH) were 5'-GTCTCTTCGGGAACTGCAAG-3'/5'-TGGGACACAGGCACTGTAGA-3' and 5'-GCTCTCTGCTCCTCCTGTTC-3'/5'-CAATACGACCAAATCCGTTG-3', respectively (forward/reverse sequence).
Quantitative real-time PCR was performed on an ABI Step One Real-time PCR system (Applied Biosystems, Foster, CA, USA) using the following program: 45 cycles of 95℃ for 10 s, 60℃ for 15 s, and 72℃ for 20 s. To normalize the amount of sample cDNA in each reaction, the Ct value of each target gene was subtracted by that of GAPDH to obtain delta Ct [14]. To calculate the fold-change in expression, the delta Ct value of the case sample was subtracted by that of the control (delta delta Ct = delta Ct case - delta Ct control).
Reverse-transcription PCR was performed similarly to quantitative real-time PCR, except that it used 35 cycles for amplification. The PCR products were separated by electrophoresis in a 2.0% agarose gel and visualized under UV lamp after ethidium bromide staining.