Departamento de Biotecnología, Universidad Autónoma Metropolitana-Unidad Iztapalapa, Mexico City 09340, Mexico.
Consejo Nacional de Ciencia y Tecnología (CONACYT), Instituto Nacional de Pediatría, Secretaría de Salud, Mexico City 04530, Mexico.
J Microbiol Methods. 2020 Mar;170:105855. doi: 10.1016/j.mimet.2020.105855. Epub 2020 Jan 28.
Transcriptomic analysis is an OMICs technology that is becoming indispensable to understand and get a complete picture of cell functioning and adaptation to the environmental cues the cell is continuously receiving. Among the techniques available to perform transcriptomics, RNA-seq is becoming the method of choice. The quality of the RNA used for the generation of cDNA libraries and subsequent sequencing is crucial for the success of the process. Good RNA-seq performance is often limited by problems such as low RNA yield and/or integrity, RNA stability, and contamination with DNA, salts or chemicals. RNA isolation from fungi usually faces these problems and is particularly sensitive to degradation due to the high RNase activity content present in many species. Here we describe the development of a robust, highly reproducible and simple RNA purification method for filamentous fungi, which combines various strategies to get fully DNA-free RNA samples of high purity and integrity without the need to use a DNase I digestion step. The obtained RNA samples complied with all required standards to be used for RNA-seq and showed an excellent performance when subjected to Illumina-HiSeq 2500.
转录组分析是一种组学技术，对于理解细胞的功能以及适应细胞不断接收到的环境信号，它变得不可或缺。在可用于进行转录组学的技术中，RNA-seq 正成为首选方法。用于生成 cDNA 文库和随后测序的 RNA 的质量对于该过程的成功至关重要。良好的 RNA-seq 性能通常受到诸如 RNA 产量和/或完整性低、RNA 稳定性以及与 DNA、盐或化学物质的污染等问题的限制。真菌的 RNA 分离通常会面临这些问题，并且由于许多物种中存在高含量的 RNase 活性，因此特别容易降解。在这里，我们描述了一种用于丝状真菌的强大、高度可重复且简单的 RNA 纯化方法的开发，该方法结合了各种策略，可获得无 DNA 的高纯度和完整性的 RNA 样品，而无需使用 DNA 酶 I 消化步骤。所获得的 RNA 样品符合用于 RNA-seq 的所有要求标准，并且在经受 Illumina-HiSeq 2500 时表现出优异的性能。
