Briefly, ribosomes were isolated and concentrated by sucrose-gradient centrifugation, and treated with a ribonuclease to remove extraneous RNA from the ribosome preparation. Next, the poly(A) portion of the RNA was isolated from the ribosomal proteins, followed by size selection. Reverse transcription of the enriched poly(A) mRNA was performed using a novel primer that contained Illumina® sequences P5 and P7 for bridge PCR/cluster generation on a flow cell and sequencing priming sites, as well as an abasic site that allows for chemical cleavage. Circligase enzyme was used to recircularize the size-selected, single-stranded (ss) DNA. Following recircularization, the ssDNA templates were relinearized using chemical methods and PCR-enriched. The templates were then sequenced on an Illumina GAII instrument.
In this study, the authors were able to compare the relative abundance of the footprinted RNA with total mRNA isolated from the cells; they determined that functional down-regulation by some miRNAs was associated with decreases in both overall mRNA abundance and ribosome loading. They also determined that the changes were of substantially smaller magnitude than corresponding changes observed in translated protein abundance. Other miRNA targets showed only modest effects, consistent with models in which miRNA-mediated regulation occurs through a combination of mechanisms.
Stadler M et al. (2012). Contributions of mRNA abundance, ribosome loading, and post- or peri-translational effects to temporal repression of C. elegans heterochronic miRNA targets. Genome Res. PMID: 22855835