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

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 mu g, 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

Study design.

<p>For comparison of extraction efficiency and RNA quality of whole blood stabilization tubes (PAXgene Blood RNA Tubes, Qiagen; Tempus Blood RNA Tubes, Life Technologies), blood was drawn from 47 probands of the LIFE and LIFE-AMI cohorts and RNA was isolated with 4 manual (white) and 2 (semi-)automated (light green) extraction kits. RNA quantity and quality was determined in 262 samples (20 extractions were lost due to handling errors). Samples from 12 probands were selected for RT and qRT-PCRs. Both steps were carried out with reagents from Qiagen or Life Technologies, respectively. Quantification of miRNA and mRNA expression was done using the ViiA7 Real-Time PCR System (Life Technologies). In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113298#pone.0113298.s001" target="_blank">Figure S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113298#pone.0113298.s004" target="_blank">Table S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113298#pone.0113298.s005" target="_blank">Table S3</a>, detailed information regarding the reaction setup for RT and qRT-PCR experiments is given. rct = reaction.</p

Summary of technical characteristics of applied RNA extraction methods from PAXgene and Tempus Blood RNA Tubes.

a<p>The time for centrifugation/vortexing, incubation steps and hands-on time was determined.</p>b<p>During centrifugation, time was used for preparation of next steps including labeling. Thus, total duration might be different from sum of single steps. Av. = Average.</p>c<p>DNase digestion was performed with RNase-Free DNase I Kit from Norgen Biotek.</p><p>Summary of technical characteristics of applied RNA extraction methods from PAXgene and Tempus Blood RNA Tubes.</p

Biological validation of investigated mRNA and miRNA transcripts in LIFE-AMI and LIFE probands.

<p>ΔCt analysis of (A) <i>beta actin</i> (<i>ACTB</i>), <i>matrix metalloproteinase 9</i> (<i>MMP9</i>) and <i>arginase 1</i> (<i>ARG1</i>) expression and (B) miR16, miR30b, miR133a and miR1 expression in in LIFE-AMI probands compared to LIFE probands depending on the RNA extraction kit (n = 6) and RT/qRT-PCR reagents from Qiagen (non-shaded bars) and Life Technologies (shaded bars), respectively. Data are given as mean and SEM.</p

Analyses of absolute Ct-values and coefficients of variation of investigated mRNAs and miRNAs.

<p>RNA from 12 probands, extracted with 6 RNA isolation kits from two collection tubes (PAXgene [P] and Tempus Blood RNA Tubes [T]) (n = 72) was reverse transcribed and analyzed by qRT-PCRs with reagents from Qiagen (Q) and Life Technologies (LT). Results of qRT-PCRs showing mean Ct-values (error bars indicate SEM) for (A) mRNA and (B) miRNA transcripts. (C,D) corresponding mean coefficients of variation (CV).</p

Quantity and quality of RNA preserved with PAXgene and Tempus Blood RNA Tubes.

<p>RNA quantity and quality were determined by OD and RNA integrity measurements. (A) RNA yield normalized to input whole blood volume (µg/g) and (B) absolute RNA recovery (µg). Significantly more RNA was recovered in samples from LIFE-AMI probands compared to LIFE probands (11.2 µg and 6.7 µg, respectively). In LIFE-AMI probands, the choice of collection tube did not affect RNA yields. In LIFE probands, higher RNA yields were recovered when using Tempus Blood RNA Tubes compared to PAXgene Blood RNA Tubes (8.3 µg and 6.5 µg, respectively). Outliers and missing values were omitted according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113298#pone.0113298.s003" target="_blank">Table S1</a>. Data are given as mean and SEM. (C) RNA samples extracted with six methods from representative LIFE and LIFE-AMI probands were analyzed on an Agilent Bioanalyzer. Please note the different scales in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113298#pone-0113298-g002" target="_blank">Figure 2C</a>. RIN = RNA integrity number.</p

Supplementary Material for: Brain Arousal Regulation in Carriers of Bipolar Disorder Risk Alleles

<br><strong><em>Objectives:</em></strong> Recent genome-wide association studies identified a number of chromosomal risk loci for bipolar disorder (BD, ‘manic-depressive illness'). According to the vigilance regulation model, the regulation of brain arousal (referred to as ‘vigilance') when assessed via EEG is an emerging biomarker linked to the pathogenesis of manic and depressive episodes. On this basis, the present study aimed to assess whether carriers of BD risk alleles differ in brain arousal regulation. <b><i>Methods:</i></b> Healthy participants of the population-based Leipzig Health Care Study (LIFE) underwent a 20-min eyes-closed resting EEG paradigm. Brain arousal was assessed applying the computer-based Vigilance Algorithm Leipzig (VIGALL). The primary sample (n = 540) was genotyped for ten of the most reliable BD risk variants, of which two qualified for replication (n = 509). <b><i>Results:</i></b> Primary sample analyses revealed Bonferroni-adjusted significance for rs1006737 in CACNA1C (encoding a calcium channel subunit), with risk allele carriers exhibiting relatively steep brain arousal declines. Further, carriers of two risk alleles of rs472913 at 1p32.1 showed generally lower brain arousal levels for the duration of the resting paradigm. However, both associations failed replication. <b><i>Conclusion:</i></b> Although our initial findings are in line with the vigilance regulation model and convincing in view of the previously reported notable role of ion channelopathies in BD, our results do not provide consistent evidence for a link between BD risk variants and brain arousal regulation. Several between-sample differences may account for this inconsistency. The molecular genetics of brain arousal regulation remain to be clarified