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

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

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

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

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

Etiology.

<p>Etiology.</p

Baseline tumor-associated parameters.

<p>(CI = confidence interval; * = statistically significant).</p

Kaplan-Meier survival analysis stratified according to the CLIP-Score (n = 352).

<p>(CLIP 0 vs. CLIP 1: p = 0.262; CLIP 1 vs. CLIP 2: p = 0.001*; CLIP 2 vs. CLIP 3: p = 0.023*; CLIP 3 vs. CLIP≥4: p = 0.005*).</p

Baseline demographic and clinical parameters.

<p>(* = statistically significant). Mo = moderate, ma = massive, part = partial, comp = complete.</p

Baseline laboratory parameters.

<p>(P* = percentile).</p