Quantifying mRNA Levels by Real-Time PCR

Real-time PCR was performed using standard methods as described [20 (link)]. The first-strand 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, Universal Master Mix (Applied Biosystems), and 10μM of forward and reverse primers in a final reaction volume of 20 μL. The data analysis software built-in with the 7300 Real-Time PCR System calculated the mRNA level of different samples. The sequences of primers used for real-time PCR reactions in mouse species are listed in Additional file 4: Table S1.