A comparative analysis of mRNA enrichment strategies and guidance for improving their efficiency

The pervasive presence of ribosomal RNA (rRNA) in total RNA poses a considerable challenge to the accurate analysis of cellular transcriptomes. In this study, we comprehensively analyze strategies for Saccharomyces cerevisiae mRNA enrichment, aiming to either separate polyadenylated RNAs or selectively remove rRNAs. Our findings reveal that a single round of mRNA enrichment in recommended conditions proves insufficient for both methods, prompting the exploration of strategies to enhance their efficiency. We show that adjusting the oligo (dT) magnetic beads-to-RNA ratio leads to significant improvement, but even better results can be achieved with two rounds of mRNA enrichment. We propose experimental conditions that reduce rRNA content in total yeast RNA to less than 10%, as confirmed by capillary electrophoresis and NG sequencing. Based on the obtained data, we selected the most time- and cost-effective option of polyadenylated RNA selection in yeast total RNA. Furthermore, we demonstrate that RNA modification with SHAPE reagent (NAI) does not interfere with the optimized mRNA enrichment protocol. These insights contribute to mRNA enrichment strategies and underscore the importance of optimizing mRNA isolation methodologies for downstream analyses.