Center for Biosignatures Discovery Automation, The Biodesign Institute, Arizona State University, Tempe, AZ 85287-6501, USA.
J Microbiol Methods. 2011 Jan;84(1):88-93. doi: 10.1016/j.mimet.2010.10.018. Epub 2010 Oct 31.
Limitation in sample quality and quantity is one of the big obstacles for applying metatranscriptomic technologies to explore gene expression and functionality of microbial communities in natural environments. In this study, several amplification methods were evaluated for whole-transcriptome amplification of deep-sea microbial samples, which are of low cell density and high impurity. The best amplification method was identified and incorporated into a complete protocol to isolate and amplify deep-sea microbial samples. In the protocol, total RNA was first isolated by a modified method combining Trizol (Invitrogen, CA) and RNeasy (QIAGEN, CA) method, amplified with a WT-Ovation™ Pico RNA Amplification System (NuGEN, CA), and then converted to double-strand DNA from single-strand cDNA with a WT-Ovation™ Exon Module (NuGEN, CA). The products from the whole-transcriptome amplification of deep-sea microbial samples were assessed first through random clone library sequencing. The BLAST search results showed that marine-based sequences are dominant in the libraries, consistent with the ecological source of the samples. The products were then used for next-generation Roche GS FLX Titanium sequencing to obtain metatranscriptome data. Preliminary analysis of the metatranscriptomic data showed good sequencing quality. Although the protocol was designed and demonstrated to be effective for deep-sea microbial samples, it should be applicable to similar samples from other extreme environments in exploring community structure and functionality of microbial communities.
样本质量和数量的限制是将宏转录组学技术应用于探索自然环境中微生物群落的基因表达和功能的主要障碍之一。在这项研究中，评估了几种扩增方法，以对低细胞密度和高杂质的深海微生物样本进行全转录组扩增。确定了最佳的扩增方法，并将其纳入完整的方案中，以分离和扩增深海微生物样本。在该方案中，首先通过结合 Trizol（Invitrogen，CA）和 RNeasy（QIAGEN，CA）方法的改良方法提取总 RNA，使用 WT-Ovation™ Pico RNA 扩增系统（NuGEN，CA）扩增，然后使用 WT-Ovation™ Exon Module（NuGEN，CA）将单链 cDNA 转化为双链 DNA。首先通过随机克隆文库测序评估深海微生物样本全转录组扩增的产物。BLAST 搜索结果表明，文库中以海洋基序列为主，与样品的生态来源一致。然后，将产物用于下一代 Roche GS FLX Titanium 测序以获得宏转录组数据。对宏转录组数据的初步分析表明测序质量良好。尽管该方案是专为深海微生物样本设计和证明有效的，但它应该适用于其他极端环境中的类似样本，以探索微生物群落的群落结构和功能。
