Comparative analysis of mRNA distribution in heavy and light polysomes in wild type and Dicer1 knockout cells

To gain a global view of translational inhibition by microRNA (miRNA), we isolated polysomes from wild type and Dicer1 knockout HCT116 human cells using sucrose gradient fractionation technique. The polysome fraction was separated into light (9mer or less) and heavy (10mer or more) subfractions. RNA samples were extracted from both subfractions and subjected to RNA-seq analysis. A general shift from light to heavy subfractions was detected for miRNA targted mRNAs. Overall design: Cytoplasmic extract was subjected to standard sucrose gradient fractionation. Light and heavy polysome fractions were identified based on the OD260 profile.