Andrzejewska-Romanowska Angelika, Tykwińska Ewa, Śledziński Paweł, Pachulska-Wieczorek Katarzyna
Department of RNA Structure and Function, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.
Sci Rep. 2025 May 23;15(1):17890. doi: 10.1038/s41598-025-02082-z.
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.
核糖体RNA（rRNA）在总RNA中的普遍存在对细胞转录组的准确分析提出了相当大的挑战。在本研究中，我们全面分析了酿酒酵母mRNA富集策略，旨在分离多聚腺苷酸化RNA或选择性去除rRNA。我们的研究结果表明，在推荐条件下进行一轮mRNA富集对这两种方法来说都证明是不够的，这促使我们探索提高其效率的策略。我们表明，调整寡聚（dT）磁珠与RNA的比例会带来显著改善，但两轮mRNA富集能取得更好的结果。我们提出了将酵母总RNA中rRNA含量降低至10%以下的实验条件，这已通过毛细管电泳和NG测序得到证实。基于所获得的数据，我们在酵母总RNA中选择了最省时且最具成本效益的多聚腺苷酸化RNA选择方案。此外，我们证明用SHAPE试剂（NAI）进行RNA修饰不会干扰优化后的mRNA富集方案。这些见解有助于mRNA富集策略，并强调了为下游分析优化mRNA分离方法的重要性。
