Gifford Scott, Satinsky Brandon, Moran Mary Ann
Department of Marine Sciences, University of Georgia, Athens, GA, USA.
Methods Mol Biol. 2014;1096:213-29. doi: 10.1007/978-1-62703-712-9_17.
The direct retrieval and sequencing of environmental RNA is emerging as a powerful technique to elucidate the in situ activities of microbial communities. Here we provide a metatranscriptomic protocol describing environmental sample collection, rRNA depletion, mRNA amplification, cDNA synthesis, and bioinformatic analysis. In addition, the preparation of internal RNA standards and their addition to the sample are discussed, providing a method by which transcript numbers can be expressed as absolute abundances in the environment and more readily compared to other biogeochemical and ecological measurements.
环境RNA的直接检索和测序正在成为一种强大的技术，用于阐明微生物群落的原位活性。在这里，我们提供了一个宏转录组学方案，描述了环境样本采集、rRNA去除、mRNA扩增、cDNA合成和生物信息学分析。此外，还讨论了内部RNA标准品的制备及其添加到样本中的方法，提供了一种可以将转录本数量表示为环境中绝对丰度的方法，并且可以更容易地与其他生物地球化学和生态学测量结果进行比较。
