Heera Rajandas, Sivachandran Parimannan, Chinni Suresh V, Mason Joanne, Croft Larry, Ravichandran Manickam, Yin Lee Su
Department of Biotechnology, Faculty of Applied Sciences, AIMST University, Semeling, 08100, Bedong, Kedah, Malaysia.
Unit of Biochemistry, Faculty of Medicine, AIMST University, Semeling, 08100, Bedong, Kedah, Malaysia.
BMC Res Notes. 2015 Dec 8;8:754. doi: 10.1186/s13104-015-1726-3.
Next-generation transcriptome sequencing (RNA-Seq) has become the standard practice for studying gene splicing, mutations and changes in gene expression to obtain valuable, accurate biological conclusions. However, obtaining good sequencing coverage and depth to study these is impeded by the difficulties of obtaining high quality total RNA with minimal genomic DNA contamination. With this in mind, we evaluated the performance of Phenol-free total RNA purification kit (Amresco) in comparison with TRI Reagent (MRC) and RNeasy Mini (Qiagen) for the extraction of total RNA of Pseudomonas aeruginosa which was grown in glucose-supplemented (control) and polyethylene-supplemented (growth-limiting condition) minimal medium. All three extraction methods were coupled with an in-house DNase I treatment before the yield, integrity and size distribution of the purified RNA were assessed. RNA samples extracted with the best extraction kit were then sequenced using the Illumina HiSeq 2000 platform.
TRI Reagent gave the lowest yield enriched with small RNAs (sRNAs), while RNeasy gave moderate yield of good quality RNA with trace amounts of sRNAs. The Phenol-free kit, on the other hand, gave the highest yield and the best quality RNA (RIN value of 9.85 ± 0.3) with good amounts of sRNAs. Subsequent bioinformatic analysis of the sequencing data revealed that 5435 coding genes, 452 sRNAs and 7 potential novel intergenic sRNAs were detected, indicating excellent sequencing coverage across RNA size ranges. In addition, detection of low abundance transcripts and consistency of their expression profiles across replicates from the same conditions demonstrated the reproducibility of the RNA extraction technique.
Amresco's Phenol-free Total RNA purification kit coupled with DNase I treatment yielded the highest quality RNAs containing good ratios of high and low molecular weight transcripts with minimal genomic DNA. These RNA extracts gave excellent non-biased sequencing coverage useful for comprehensive total transcriptome sequencing and analysis. Furthermore, our findings would be useful for those interested in studying both coding and non-coding RNAs from precious bacterial samples cultivated in growth-limiting condition, in a single sequencing run.
新一代转录组测序（RNA-Seq）已成为研究基因剪接、突变及基因表达变化以获取有价值、准确生物学结论的标准方法。然而，由于难以获得高质量且基因组DNA污染最小的总RNA，阻碍了获得用于研究这些方面的良好测序覆盖度和深度。考虑到这一点，我们评估了无酚总RNA纯化试剂盒（Amresco）与TRI试剂（MRC）和RNeasy Mini（Qiagen）相比，用于提取在补充葡萄糖（对照）和补充聚乙烯（生长限制条件）的基本培养基中生长的铜绿假单胞菌总RNA的性能。在评估纯化RNA的产量、完整性和大小分布之前，所有三种提取方法均与内部DNase I处理相结合。然后使用Illumina HiSeq 2000平台对用最佳提取试剂盒提取的RNA样本进行测序。
TRI试剂的产量最低，富含小RNA（sRNA），而RNeasy产生的高质量RNA产量适中，sRNA含量微量。另一方面，无酚试剂盒产生的产量最高，RNA质量最佳（RIN值为9.85±0.3），且有大量sRNA。对测序数据的后续生物信息学分析表明，检测到5435个编码基因、452个sRNA和7个潜在的新型基因间sRNA，表明在RNA大小范围内具有出色的测序覆盖度。此外，低丰度转录本的检测及其在相同条件下重复样本中表达谱的一致性证明了RNA提取技术的可重复性。
Amresco的无酚总RNA纯化试剂盒与DNase I处理相结合，产生了质量最高的RNA，其中包含高分子量和低分子量转录本的良好比例，且基因组DNA最少。这些RNA提取物提供了出色的无偏测序覆盖度，可用于全面的总转录组测序和分析。此外，我们的研究结果对于那些有兴趣在单次测序运行中研究在生长限制条件下培养的珍贵细菌样本中的编码和非编码RNA的人将是有用的。
