Quantitative real-time RT–PCR in tissue, cells, and polysome fractionsTotal RNA was extracted using Trizol reagent and the Qiagen RNeasy mini kit or micro kit, as previously described (Yip et al., 2009). First-strand cDNA was generated using the iScript cDNA synthesis kit (Biorad). Quantitative PCR was performed to measure mouse Eif4g3, Eif4g1, Casp3, Deaf1, Ins2, Fgb, Ela, Ppy, Tyr, Ambp, Gapdh, and Actb mRNA levels, and human EIF4G3, EIF4G1, CASP3 and ACTB, and GAPDH mRNA levels. cDNA was preamplified using the Taqman PreAmp Mastermix (Applied Biosystems) prior to QPCR for Ins2 and for gene expression measured in polysome fractions and LNSC subsets. For all other experiments, cDNA was not pre-amplified. qPCR assays were performed using the 7900HT Fast Real Time PCR System (Applied Biosystems), Taqman Gene Expression Arrays (Applied Biosystems), and SsoFast Probes Supermix (Biorad). For human CASP3 measurements, Quantitect primers (Qiagen) and SsoFast EvaGreen Supermix (Biorad) were used. The comparative Ct method for relative quantification (ΔΔCt) was used, and expression was normalized with housekeeping gene expression.