Polysome Fractionation and Quantitative RT-PCR

Polysome fractionation and RNA preparation were carried out as previous (Castelli et al., 2011 (link)) with the following modifications. Fifteen fractions were collected across the gradient into two volumes Trizol (Life Technologies). Four nanograms luciferase control RNA (Promega) was spiked into each fraction, and then the RNA was extracted, precipitated, and resuspended in diethyl-pyrocarbonate-treated water. The RNA was converted to cDNA using a Protoscript M-MuLV Taq RT-PCR kit (New England Biolabs), and quantitative RT-PCR (qRT-PCR) was performed with the CFx Connect Real-Time system with iTaq Universal SYBR Green Supermix (Bio-Rad). Samples were run in triplicate and normalized to luciferase RNA, and the fold change was calculated using 2^−ΔCt for each tested RNA. From this, the percentage of the test RNA in each fraction was calculated.