Real-Time PCR for Quantifying mRNA Expression

Real-time PCR was performed using standard methods as previously described^{14 (link)}. The first-strand complementary DNA (cDNA) was generated by reverse transcription with Oligo (dT) primer (Roche). To quantify the mRNA levels using real-time PCR, aliquots of first-stranded cDNA were amplified with gene-specific primers and Power SYBR Green PCR Master Mix (Bio-Rad) using a Step-1 Real-Time PCR System (Applied Biosystems). The PCR reactions contained 1 μg of cDNA (except the cDNA for the IP, for which 5% of the cDNA was used for each gene examined), Universal Master Mix (Applied Biosystems), and 10 μM of forward and reverse primers in a final reaction volume of 20 μL. The mRNA level of different samples was calculated by the data analysis software built in with the 7300 Real-Time PCR System. For RIP-real-time PCR, cDNA from IP and input was used and IP samples were normalized to Input samples. The Sequences of primers are provided in Supplementary Methods.