Chu Candice P, Nabity Mary B
Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas.
Vet Clin Pathol. 2019 Jun;48(2):310-319. doi: 10.1111/vcp.12743. Epub 2019 May 11.
Small RNA sequencing (RNA-seq) of biofluids is challenging due to the relative scarcity of microRNAs (miRNAs), limited sample volumes, and the lack of a gold standard isolation method. Additionally, few comparisons exist for the RNA isolation and sequencing methods of biofluids.
We aimed to compare the performance of six commercial RNA isolation kits and two library preparation methods for small RNA-seq using canine serum and urine.
Serum and urine were collected from seven dogs with protein-losing nephropathy, and the samples were pooled. Total RNA from serum (2 mL) and urine (10 mL) was isolated in triplicate using three methods each for serum (Zymo Direct-zol, mirVana PARIS, miRCURY Biofluids) and urine (Qiagen exoRNeasy, Norgen Urine Exosome, miRCURY Exosome). For each sample type, the two kits yielding the highest RNA concentration were selected, and small RNA-seq was performed using TruSeq and NEXTflex library preparations. Data were analyzed by CPSS 2.0 and DESeq2.
For serum, Zymo Direct-zol combined with NEXTflex was the only combination that enabled successful library preparation, while for urine, Qiagen exoRNeasy combined with NEXTflex outperformed other combinations for detecting miRNAs. The total number of miRNAs detected in serum and urine was 198 and up to 115, respectively. miRNA expression in serum was distinct from urine. Furthermore, the library preparation method introduced a higher variation of urine results than the RNA isolation method.
Different isolation and library preparation methods show significant differences in miRNA results that could affect biomarker discovery. Small RNA-seq provides an unbiased, global assessment to compare these methods in canine biofluids.
由于微RNA（miRNA）相对稀少、样本量有限以及缺乏金标准分离方法，生物流体的小RNA测序（RNA-seq）具有挑战性。此外，对于生物流体的RNA分离和测序方法，很少有比较研究。
我们旨在比较六种商业RNA分离试剂盒和两种文库制备方法在使用犬血清和尿液进行小RNA-seq时的性能。
从七只患有失蛋白性肾病的犬只收集血清和尿液，并将样本合并。使用三种方法分别从血清（2 mL）和尿液（10 mL）中提取总RNA，血清提取方法为Zymo Direct-zol、mirVana PARIS、miRCURY Biofluids，尿液提取方法为Qiagen exoRNeasy、Norgen Urine Exosome、miRCURY Exosome，每种方法重复三次。对于每种样本类型，选择产生最高RNA浓度的两种试剂盒，并使用TruSeq和NEXTflex文库制备方法进行小RNA-seq。数据通过CPSS 2.0和DESeq2进行分析。
对于血清，Zymo Direct-zol与NEXTflex组合是唯一能够成功制备文库的组合，而对于尿液，Qiagen exoRNeasy与NEXTflex组合在检测miRNA方面优于其他组合。血清和尿液中检测到的miRNA总数分别为198个和多达115个。血清中的miRNA表达与尿液不同。此外，文库制备方法对尿液结果的影响比RNA分离方法更大。
不同的分离和文库制备方法在miRNA结果上显示出显著差异，这可能会影响生物标志物的发现。小RNA-seq提供了一种无偏倚的全局评估方法，用于比较犬生物流体中的这些方法。
