Quantitative PCR Protocol for Gene Expression

Total RNA was isolated from tissue samples using the miRNA isolation kit (Qiagen, Germantown, MD) according to the protocol provided by the manufacturer. The cDNA was synthesized using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). Quantitative PCR was performed using gene-specific primers (Supplemental Table S3) and the Roche SYBR green master mix. Samples were run on a LightCycler 480 II system (Roche, Pleasanton, CA) and analyzed using the Roche LightCycler 1.5.0 software^{26 (link)}. All qPCR targets were quantified based on standard curves ran on the sample plate. The mRNA levels of specific genes were normalized to the mRNA levels of housekeeping genes of the same sample. The mRNA levels of housekeeping genes eukaryotic translation initiation factor 2A (Eif2a, Fig. 2a), glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Fig. 2d,g), ribosomal protein L13a (Rpl13a, Fig. 3d), and β2 microglobulin (B2M, Supplemental Fig. S2) were used for normalization.