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

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

GWAS results for amino acids (a) and acylcarnitines (b) in whole blood.

<p>Manhattan plots of the genome-wide association analysis for metabolic phenotypes in 2,107 individuals of the LIFE-Heart cohort. Results are presented separately for 36 acylcarnitines (including free and total carnitine) and 26 amino acids. Results for metabolite ratios are omitted. The horizontal line represents a p-value = 1.0x10^-7, which was the cutoff used for inclusion of identified associations in the replication state.</p

Results of SNP-metabolite association analyses.

<p>Table includes all validated loci of our analysis. Validation is based on either successful replications in the Sorbs or by additional published evidence. The latter applies for two loci (#4 and #14) where association of lead-SNPs did not replicate in the Sorbs cohort. For each locus, nearby genes, independently associated SNPs, associated metabolites and statistics for the strongest association between them are shown (Beta estimators, corresponding standard errors and p-values). We also present the results of replication analysis and published evidence. Six loci with no corresponding published genetic variants were considered as “novel”.</p><p>¹SNP with strongest association in the discovery cohort is presented in bold;</p><p>²Distance of SNPs to genes in kB in parentheses;</p><p>³Metabolite with strongest association in the discovery cohort is presented in bold. p-value Sorbs: best p-value of SNPs in Sorbs corresponding to the lead-SNP and metabolite of discovery cohort,</p><p>⁴Replication was successful for ratio Q14:Arg/Orn, only, hence, we report here on association with Q14:Arg/Orn</p><p>Results of SNP-metabolite association analyses.</p

Results of eQTL analysis of validated loci.

<p>¹SNP with strongest metabolite association is presented in <b>bold</b> while SNP with strongest eQTL was marked with an asterisk<b>*</b></p><p>²Gene with strongest association is presented in <b>bold</b></p><p>³A q-value<5% was considered as significant, i.e. FDR is controlled at 5%.</p><p>⁴These genes are located on the same chromosome as the lead-SNPs at distances larger than 1Mb</p><p>Results of eQTL analysis of validated loci.</p

Network of discovered loci, eQTLs and metabolites.

<p>Significant relationships between genetic loci (top SNPs), gene-expression in PBMCs and metabolite levels in whole blood are displayed. Line thickness corresponds to amount of explained variance (Lightblue = genetic loci without triangles, darkblue = genetic loci with triangles, lightgreen = cis-regulated genes, darkgreen = trans-regulated genes, light orange = raw metabolites, darkorange = metabolite ratios). An interactive html-document document of the network can be found in the supplement material.</p

eQTL map of mQTL loci.

<p>We analysed the top-SNPs of our mQTL analysis regarding association with gene-expression levels. A total of 54 top-SNPs were correlated with 28,295 probe expressions. Expression probes of auto- and gonosomes were analysed, while SNPs were restricted to autosomes. X-axis represents physical position of SNPs. Y-axis represents the physical position of the start of the regulated transcript. Points located on the diagonal line relate to cis-effects, while other points relate to trans-effects. Associations with FDR = 5% are highlighted. Trans-eQTLs with p-values ≤ 0.001 are also shown. Size of points represents the strength of association. Colors of points and gray shadings indicate distinct chromosomes. An interactive html version of this map allowing exploration of the results is provided as supplemental <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005510#pgen.1005510.s007" target="_blank">S7 Fig</a>.</p