用于分析mRNA和miRNA谱的全血RNA保存管与新一代RNA提取试剂盒的比较。
Comparison of whole blood RNA preservation tubes and novel generation RNA extraction kits for analysis of mRNA and MiRNA profiles.
作者信息
Häntzsch Madlen, Tolios Alexander, Beutner Frank, Nagel Dorothea, Thiery Joachim, Teupser Daniel, Holdt Lesca M
机构信息
LIFE - Leipzig Research Center for Civilization Diseases, University Leipzig, Leipzig, Germany; Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany.
Institute of Laboratory Medicine, Ludwig-Maximilians-University Munich, Munich, Germany.
出版信息
PLoS One. 2014 Dec 3;9(12):e113298. doi: 10.1371/journal.pone.0113298. eCollection 2014.
BACKGROUND
Whole 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.
METHODS
3 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.
RESULTS
RNA 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, p<0.001) followed by the choice of blood collection tube. (Semi-)automation reduced overall RNA extraction time but did not generally reduce hands-on-time. RNA yields and quality were comparable between manual and automated extraction protocols. mRNA expression was not affected by collection tubes and RNA extraction kits but by RT/qPCR reagents with exception of the Norgen Kit II, which led to mRNA depletion. For miRNAs, expression differences related to collection tubes (miR30b), RNA isolation (Norgen Kit II), and RT/qRT reagents (miR133a) were observed.
CONCLUSION
We 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使用PAXgene（Qiagen公司）或Tempus（赛默飞世尔科技公司）采血管进行。在此，我们比较了6种新一代RNA分离方案在RNA数量、质量以及mRNA和miRNA回收率方面的差异。
方法
从LIFE研究的参与者中收集了3支PAXgene采血管和3支Tempus采血管，其中有急性心肌梗死（AMI）的参与者12例，无AMI的参与者35例。使用Qiagen公司（PAXgene Blood miRNA试剂盒）、赛默飞世尔科技公司（用于稳定采血管的MagMAX RNA分离试剂盒）和Norgen Biotek公司（Norgen Preserved Blood RNA纯化试剂盒I和试剂盒II）的4种手动方案，以及QIAsymphony（Qiagen公司）和MagMAX Express - 96磁性粒子处理器（赛默飞世尔科技公司）上的2种（半）自动化方案提取RNA。测定RNA的数量和质量。为进行生物学验证，对用所有6种试剂盒从12名代表性先证者中提取的RNA（n = 72）进行逆转录，并分析AMI后显示有变化的mRNA（基质金属蛋白酶9、精氨酸酶1）和miRNA（miR133a、miR1）。
结果
使用Norgen试剂盒I和Tempus采血管时RNA产量最高，使用Norgen试剂盒II和PAXgene采血管时RNA产量最低。疾病状态是RNA产量的第二大主要决定因素（LIFE - AMI组为11.2μg，LIFE组为6.7μg，p<0.001），其次是采血管的选择。（半）自动化减少了总体RNA提取时间，但一般并未减少实际操作时间。手动和自动化提取方案之间的RNA产量和质量相当。mRNA表达不受采血管和RNA提取试剂盒的影响，但除Norgen试剂盒II导致mRNA耗竭外，受RT/qPCR试剂的影响。对于miRNA，观察到与采血管（miR30b）、RNA分离（Norgen试剂盒II）和RT/qRT试剂（miR133a）相关的表达差异。
结论
我们证明新一代RNA分离试剂盒在RNA回收率方面存在显著差异，并且会影响miRNA而非mRNA的表达谱。
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