Institut des Sciences de l'Evolution, CNRS UMR 5554, Université Montpellier 2, Place E Bataillon, 34095 Montpellier, France.
Mol Ecol Resour. 2011 Jul;11(4):650-61. doi: 10.1111/j.1755-0998.2011.03010.x. Epub 2011 Apr 12.
Next Generation Sequencing technologies (NGS) are rapidly invading many evolutionary and ecological fields, such as phylogenomics, molecular evolution, population genomics and molecular ecology. Among the potential targets of NGS is transcriptome sequencing, a fast and relatively cheap way to generate massive amounts of coding sequence data, offering promising perspectives for the analysis of molecular diversity in the wild. A number of molecular ecology research groups therefore may switch from DNA-based to RNA-based typing in the near future. Sample preparation from natural populations, however, requires specific care and protocols when RNA is the target. Furthermore, NGS sequencing of transcriptome requires high amount of good-quality RNA. Here we present the results of RNA extraction experiments from various samples of 39 animal species caught in the wild. We compared tissue preparation and storage conditions, evaluated and improved standard RNA extraction protocols, and achieved RNA yield and quality suitable for NGS in all cases. We derive general guidelines for the production of ready-to-sequence RNA in nonmodel animals sampled in the field.
下一代测序技术（NGS）正在迅速入侵许多进化和生态领域，如系统发生基因组学、分子进化、群体基因组学和分子生态学。NGS 的潜在目标之一是转录组测序，这是一种快速且相对廉价的方法，可以生成大量的编码序列数据，为分析野外的分子多样性提供了有前途的视角。因此，许多分子生态学研究小组可能会在不久的将来从基于 DNA 的分型转向基于 RNA 的分型。然而，当 RNA 是目标时，从自然种群中提取样本需要特别的注意和特定的方案。此外，转录组的 NGS 测序需要大量高质量的 RNA。在这里，我们展示了从野外捕获的 39 种动物的各种样本中提取 RNA 的实验结果。我们比较了组织准备和储存条件，评估和改进了标准 RNA 提取方案，并在所有情况下都获得了适合 NGS 的 RNA 产量和质量。我们为在野外采样的非模式动物生成可随时进行测序的 RNA 提供了一般指导方针。
