O'Connell Aoife, Fedorova Alla D, O'Connor Patrick B F, Zhdanov Alexander V, Baranov Pavel V, Loughran Gary, Andreev Dmitry E
School of Biochemistry and Cell Biology, University College Cork, Cork, Ireland.
EIRNA Bio Ltd, Bioinnovation Hub, Food Science & Technology Building, College Road, Cork, Ireland.
Genome Biol. 2025 Aug 11;26(1):241. doi: 10.1186/s13059-025-03651-1.
Ribosome profiling is a powerful technique used to study gene expression on a transcriptome-wide scale. It involves sequencing of mRNA fragments protected by ribosomes from ribonuclease digestion. The initial steps commonly involve cell lysis followed by centrifugation and ribonuclease digestion. We find that centrifugation depletes 329 translated mRNAs in HEK293T cells. Many of these mRNAs encode cytoskeleton proteins. This suggests that the expression of a subset of mRNAs may be significantly underestimated in most ribosome profiling experiments. We show that omitting the centrifugation step after cell lysis can resolve this issue.
核糖体谱分析是一种用于在全转录组范围内研究基因表达的强大技术。它涉及对受核糖体保护而免受核糖核酸酶消化的mRNA片段进行测序。最初的步骤通常包括细胞裂解，随后进行离心和核糖核酸酶消化。我们发现离心会使HEK293T细胞中329种已翻译的mRNA减少。这些mRNA中有许多编码细胞骨架蛋白。这表明在大多数核糖体谱分析实验中，一部分mRNA的表达可能被显著低估。我们表明，在细胞裂解后省略离心步骤可以解决这个问题。
