Open AccessTargeted Profiling of RNA Translation
Author(s) -
Li Ben B.,
Qian Changli,
Roberts Thomas M.,
Zhao Jean J.
Publication year - 2019
Publication title -
current protocols in molecular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.533
H-Index - 42
eISSN - 1934-3647
pISSN - 1934-3639
DOI - 10.1002/cpmb.71
Subject(s) - cycloheximide , biology , translation (biology) , messenger rna , rna , reverse transcriptase , computational biology , ribosomal rna , gene , complementary dna , transcription (linguistics) , protein biosynthesis , microbiology and biotechnology , genetics , linguistics , philosophy
Abstract This unit describes a reverse transcription‐quantitative PCR (RT‐qPCR)–based method for gene‐targeted measurement of RNA translation levels. The method includes washing and lysing cells with a buffer containing cycloheximide to enrich ribosomal accumulation at translation initiation sites (TIS), followed by enzymatic treatment to generate ribosomal footprints, reverse transcription targeted towards TIS of specific transcripts of interest to generate complementary DNA (cDNA), and qPCR to measure the abundance of these footprints. This method enables time‐ and cost‐effective assessment of changes in translation levels across focused panels of genes and across numerous samples. © 2018 by John Wiley & Sons, Inc.