Department of Biology and Chemistry, City University of Hong Kong, Hong Kong SAR.
PLoS One. 2013 Sep 20;8(9):e74286. doi: 10.1371/journal.pone.0074286. eCollection 2013.
RNA-sequencing is a powerful tool in studying RNomics. However, the highly abundance of ribosomal RNAs (rRNA) and transfer RNA (tRNA) have predominated in the sequencing reads, thereby hindering the study of lowly expressed genes. Therefore, rRNA depletion prior to sequencing is often performed in order to preserve the subtle alteration in gene expression especially those at relatively low expression levels. One of the commercially available methods is to use DNA or RNA probes to hybridize to the target RNAs. However, there is always a concern with the non-specific binding and unintended removal of messenger RNA (mRNA) when the same set of probes is applied to different organisms. The degree of such unintended mRNA removal varies among organisms due to organism-specific genomic variation. We developed a computer-based method to design probes to deplete rRNA in an organism-specific manner. Based on the computation results, biotinylated-RNA-probes were produced by in vitro transcription and were used to perform rRNA depletion with subtractive hybridization. We demonstrated that the designed probes of 16S rRNAs and 23S rRNAs can efficiently remove rRNAs from Mycobacterium smegmatis. In comparison with a commercial subtractive hybridization-based rRNA removal kit, using organism-specific probes is better in preserving the RNA integrity and abundance. We believe the computer-based design approach can be used as a generic method in preparing RNA of any organisms for next-generation sequencing, particularly for the transcriptome analysis of microbes.
RNA 测序是研究 RNA 组学的强大工具。然而，核糖体 RNA(rRNA)和转移 RNA(tRNA)的高度丰度在测序reads 中占主导地位，从而阻碍了低表达基因的研究。因此，在测序前通常会进行 rRNA 耗竭，以保留基因表达的细微变化，特别是那些表达水平相对较低的基因。一种商业上可用的方法是使用 DNA 或 RNA 探针与靶 RNA 杂交。然而，当同一组探针应用于不同的生物体时，总会存在非特异性结合和意外去除信使 RNA(mRNA)的问题。由于生物体特异性基因组变异，这种意外去除 mRNA 的程度在不同生物体之间有所不同。我们开发了一种基于计算机的方法来设计探针，以特异性方式耗竭 rRNA。根据计算结果，通过体外转录产生了生物素化 RNA 探针，并用于通过减法杂交进行 rRNA 耗竭。我们证明了设计的 16S rRNA 和 23S rRNA 探针能够有效地从耻垢分枝杆菌中去除 rRNA。与商业的基于减法杂交的 rRNA 去除试剂盒相比，使用生物体特异性探针在保持 RNA 完整性和丰度方面更好。我们相信基于计算机的设计方法可以用作为下一代测序准备任何生物体的 RNA 的通用方法，特别是用于微生物的转录组分析。
