Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06519, USA.
Yale Center for Genome Analysis, Yale University School of Medicine, New Haven, Connecticut 06519, USA.
RNA. 2024 May 16;30(6):728-738. doi: 10.1261/rna.079761.123.
Transcriptomics analyses play pivotal roles in understanding the complex regulatory networks that govern cellular processes. The abundance of rRNAs, which account for 80%-90% of total RNA in eukaryotes, limits the detection and investigation of other transcripts. While mRNAs and long noncoding RNAs have poly(A) tails that are often used for positive selection, investigations of poly(A) RNAs, such as circular RNAs, histone mRNAs, and small RNAs, typically require the removal of the abundant rRNAs for enrichment. Current approaches to deplete rRNAs for downstream molecular biology investigations are hampered by restrictive RNA input masses and high costs. To address these challenges, we developed RNA emoval by NaseH (rRRR), a method to efficiently deplete rRNAs from a wide range of human, mouse, and rat RNA inputs and of varying qualities at a cost 10- to 20-fold cheaper than other approaches. We used probe-based hybridization and enzymatic digestion to selectively target and remove rRNA molecules while preserving the integrity of non-rRNA transcripts. Comparison of rRRR to two commercially available approaches showed similar rRNA depletion efficiencies and comparable off-target effects. Our developed method provides researchers with a valuable tool for investigating gene expression and regulatory mechanisms across a wide range of biological systems at an affordable price that increases the accessibility for researchers to enter the field, ultimately advancing our understanding of cellular processes.
转录组学分析在理解控制细胞过程的复杂调控网络方面发挥着关键作用。真核生物中 rRNA 的丰度占总 RNA 的 80%-90%，这限制了对其他转录本的检测和研究。虽然 mRNAs 和长非编码 RNA 具有多聚（A）尾，通常用于阳性选择，但对 poly(A) RNA 的研究，如环状 RNA、组蛋白 mRNA 和小 RNA，通常需要去除丰富的 rRNA 进行富集。目前用于下游分子生物学研究的 rRNA 去除方法受到 RNA 输入质量限制和高成本的阻碍。为了解决这些挑战，我们开发了 NaseH 介导的 RNA 去除（rRRR）方法，该方法可以高效地从广泛的人类、小鼠和大鼠 RNA 输入中去除 rRNA，并且可以处理不同质量的 RNA，成本比其他方法低 10-20 倍。我们使用基于探针的杂交和酶消化来选择性地靶向和去除 rRNA 分子，同时保持非 rRNA 转录本的完整性。rRRR 与两种商业上可用的方法进行比较，显示出相似的 rRNA 耗竭效率和可比的脱靶效应。我们开发的方法为研究人员提供了一种有价值的工具，可用于在广泛的生物系统中研究基因表达和调控机制，价格实惠，增加了研究人员进入该领域的机会，最终推进我们对细胞过程的理解。
