Determination of enrichment factors for modified RNA in MeRIP experiments

Abstract

In the growing field of RNA modification, precipitation techniques using antibodies play an important role. However, little is known about their specificities and protocols are missing to assess their effectiveness. Here we present a method to assess enrichment factors after MeRIP-type pulldown experiments, here exemplified with a commercial antibody against N6-methyladenosine (m⁶A). Testing different pulldown and elution conditions, we measure enrichment factors of 4-5 using m⁶A-containing mRNAs against an unmodified control of identical sequence. Both types of mRNA carry ³²P labels at different nucleotides, allowing their relative quantification in a mixture after digestion to nucleotides, separation by TLC and quantitative phosphorimaging of the labels.

Keywords: Antibody; M(6)A/N6-methyladenosine; MeRIP; RNA modification; TLC.