Comparison of Whole Blood RNA Preservation Tubes and Novel Generation RNA Extraction Kits for Analysis of mRNA and MiRNA Profiles

BackgroundWhole blood expression profiling is frequently performed using PAXgene (Qiagen) or Tempus (Life Technologies) tubes. Here, we compare 6 novel generation RNA isolation protocols with respect to RNA quantity, quality and recovery of mRNA and miRNA.Methods3 PAXgene and 3 Tempus Tubes were collected from participants of the LIFE study with (n = 12) and without (n = 35) acute myocardial infarction (AMI). RNA was extracted with 4 manual protocols from Qiagen (PAXgene Blood miRNA Kit), Life Technologies (MagMAX for Stabilized Blood Tubes RNA Isolation Kit), and Norgen Biotek (Norgen Preserved Blood RNA Purification Kit I and Kit II), and 2 (semi-)automated protocols on the QIAsymphony (Qiagen) and MagMAX Express-96 Magnetic Particle Processor (Life Technologies). RNA quantity and quality was determined. For biological validation, RNA from 12 representative probands, extracted with all 6 kits (n = 72), was reverse transcribed and mRNAs (matrix metalloproteinase 9, arginase 1) and miRNAs (miR133a, miR1), shown to be altered by AMI, were analyzed.ResultsRNA yields were highest using the Norgen Kit I with Tempus Tubes and lowest using the Norgen Kit II with PAXgene. The disease status was the second major determinant of RNA yields (LIFE-AMI 11.2 vs. LIFE 6.7 µg, pConclusionWe demonstrate that novel generation RNA isolation kits significantly differed with respect to RNA recovery and affected miRNA but not mRNA expression profiles.

背景：全血表达谱分析通常采用PAXgene采血管（Qiagen）或Tempus采血管（Life Technologies）完成。本研究针对6种新一代RNA分离方案，对比其RNA产量、质量以及信使RNA（mRNA）和微小RNA（miRNA）的回收效率。方法：从LIFE研究的受试者中采集3份PAXgene采血管与3份Tempus采血管，其中12例合并急性心肌梗死（acute myocardial infarction, AMI），35例未罹患该病。采用4种手动分离方案提取RNA：Qiagen的PAXgene Blood miRNA Kit、Life Technologies的MagMAX for Stabilized Blood Tubes RNA Isolation Kit、Norgen Biotek的Norgen Preserved Blood RNA Purification Kit I与Kit II；同时采用2种半自动化提取方案：Qiagen的QIAsymphony系统以及Life Technologies的MagMAX Express-96磁珠处理器。随后对RNA的产量与质量进行检测与评估。为开展生物学验证，对使用全部6种试剂盒提取的12名代表性受试者的RNA样本（共72份）进行逆转录，随后分析经证实受AMI调控的mRNA标志物（基质金属蛋白酶9、精氨酸酶1）及miRNA标志物（miR133a、miR1）的表达水平。结果：采用Norgen Kit I结合Tempus采血管时，RNA产量最高；而采用Norgen Kit II结合PAXgene采血管时，RNA产量最低。疾病状态是影响RNA产量的第二大关键因素（LIFE-AMI组为11.2 µg，LIFE对照组为6.7 µg，p<0.001）。结论：本研究证实，新一代RNA分离试剂盒在RNA回收效率上存在显著差异，且会显著改变miRNA表达谱，但对mRNA表达谱无明显影响。