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

Genetic association of objective sleep phenotypes with a functional polymorphism in the neuropeptide S receptor gene

Background: The neuropeptide S receptor (NPSR1) and its ligand neuropeptide S (NPS) have received increased attention in the last few years, as both establish a previously unknown system of neuromodulation. Animal research studies have suggested that NPS may be involved in arousal/wakefulness and may also have a crucial role in sleep regulation. The single nucleotide polymorphism (SNP) rs324981 in NPSR1 has begun to shed light on a function of the NPS-system in human sleep regulation. Due to an amino acid exchange, the T-allele leads to an increased sensitivity of the NPSR1. In the only genomewide association study to date on circadian sleep parameters in humans, an association was found between rs324981 and regular bedtime. However, the sleep parameters in this study were only measured by self-rating. Therefore, our study aimed to replicate these findings using an objective measure of sleep. Methods: The study included n = 393 white subjects (62–79 years) who participated in an actigraphic assessment for determining sleep duration, rest duration, sleep onset, rest onset and sleep onset latency. Genotyping of the SNP rs324981 was performed using the TaqMan OpenArray System. Results: The genotype at rs324981 was not significantly associated with rest onset (bedtime) or sleep onset (p = .146 and p = .199, respectively). However, the SNP showed a significant effect on sleep- and rest duration (p = .007 and p = .003, respectively). Subjects that were homozygous for the minor T-allele had a significantly decreased sleep- and rest duration compared to A-allele carriers. Conclusion: The results of this study indicate that the sleep pattern in humans is influenced by the NPS-system. However, the previously reported association between bedtime and rs324981 could not be confirmed. The current finding of decreased sleep duration in T/T allele carriers is in accordance with studies in rodents reporting similar results after NPS application.:Background; Methods; Results; Conclusion

Genetic association of objective sleep phenotypes with a functional polymorphism in the neuropeptide S receptor gene.

BACKGROUND: The neuropeptide S receptor (NPSR1) and its ligand neuropeptide S (NPS) have received increased attention in the last few years, as both establish a previously unknown system of neuromodulation. Animal research studies have suggested that NPS may be involved in arousal/wakefulness and may also have a crucial role in sleep regulation. The single nucleotide polymorphism (SNP) rs324981 in NPSR1 has begun to shed light on a function of the NPS-system in human sleep regulation. Due to an amino acid exchange, the T-allele leads to an increased sensitivity of the NPSR1. In the only genome-wide association study to date on circadian sleep parameters in humans, an association was found between rs324981 and regular bedtime. However, the sleep parameters in this study were only measured by self-rating. Therefore, our study aimed to replicate these findings using an objective measure of sleep. METHODS: The study included n = 393 white subjects (62-79 years) who participated in an actigraphic assessment for determining sleep duration, rest duration, sleep onset, rest onset and sleep onset latency. Genotyping of the SNP rs324981 was performed using the TaqMan OpenArray System. RESULTS: The genotype at rs324981 was not significantly associated with rest onset (bedtime) or sleep onset (p = .146 and p = .199, respectively). However, the SNP showed a significant effect on sleep- and rest duration (p = .007 and p = .003, respectively). Subjects that were homozygous for the minor T-allele had a significantly decreased sleep- and rest duration compared to A-allele carriers. CONCLUSION: The results of this study indicate that the sleep pattern in humans is influenced by the NPS-system. However, the previously reported association between bedtime and rs324981 could not be confirmed. The current finding of decreased sleep duration in T/T allele carriers is in accordance with studies in rodents reporting similar results after NPS application

Genetic association of objective sleep phenotypes with a functional polymorphism in the neuropeptide S receptor gene

Background: The neuropeptide S receptor (NPSR1) and its ligand neuropeptide S (NPS) have received increased attention in the last few years, as both establish a previously unknown system of neuromodulation. Animal research studies have suggested that NPS may be involved in arousal/wakefulness and may also have a crucial role in sleep regulation. The single nucleotide polymorphism (SNP) rs324981 in NPSR1 has begun to shed light on a function of the NPS-system in human sleep regulation. Due to an amino acid exchange, the T-allele leads to an increased sensitivity of the NPSR1. In the only genomewide association study to date on circadian sleep parameters in humans, an association was found between rs324981 and regular bedtime. However, the sleep parameters in this study were only measured by self-rating. Therefore, our study aimed to replicate these findings using an objective measure of sleep. Methods: The study included n = 393 white subjects (62–79 years) who participated in an actigraphic assessment for determining sleep duration, rest duration, sleep onset, rest onset and sleep onset latency. Genotyping of the SNP rs324981 was performed using the TaqMan OpenArray System. Results: The genotype at rs324981 was not significantly associated with rest onset (bedtime) or sleep onset (p = .146 and p = .199, respectively). However, the SNP showed a significant effect on sleep- and rest duration (p = .007 and p = .003, respectively). Subjects that were homozygous for the minor T-allele had a significantly decreased sleep- and rest duration compared to A-allele carriers. Conclusion: The results of this study indicate that the sleep pattern in humans is influenced by the NPS-system. However, the previously reported association between bedtime and rs324981 could not be confirmed. The current finding of decreased sleep duration in T/T allele carriers is in accordance with studies in rodents reporting similar results after NPS application.:Background; Methods; Results; Conclusion

Genetic association of objective sleep phenotypes with a functional polymorphism in the neuropeptide S receptor gene

Background: The neuropeptide S receptor (NPSR1) and its ligand neuropeptide S (NPS) have received increased attention in the last few years, as both establish a previously unknown system of neuromodulation. Animal research studies have suggested that NPS may be involved in arousal/wakefulness and may also have a crucial role in sleep regulation. The single nucleotide polymorphism (SNP) rs324981 in NPSR1 has begun to shed light on a function of the NPS-system in human sleep regulation. Due to an amino acid exchange, the T-allele leads to an increased sensitivity of the NPSR1. In the only genomewide association study to date on circadian sleep parameters in humans, an association was found between rs324981 and regular bedtime. However, the sleep parameters in this study were only measured by self-rating. Therefore, our study aimed to replicate these findings using an objective measure of sleep. Methods: The study included n = 393 white subjects (62–79 years) who participated in an actigraphic assessment for determining sleep duration, rest duration, sleep onset, rest onset and sleep onset latency. Genotyping of the SNP rs324981 was performed using the TaqMan OpenArray System. Results: The genotype at rs324981 was not significantly associated with rest onset (bedtime) or sleep onset (p = .146 and p = .199, respectively). However, the SNP showed a significant effect on sleep- and rest duration (p = .007 and p = .003, respectively). Subjects that were homozygous for the minor T-allele had a significantly decreased sleep- and rest duration compared to A-allele carriers. Conclusion: The results of this study indicate that the sleep pattern in humans is influenced by the NPS-system. However, the previously reported association between bedtime and rs324981 could not be confirmed. The current finding of decreased sleep duration in T/T allele carriers is in accordance with studies in rodents reporting similar results after NPS application.:Background; Methods; Results; Conclusion

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

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

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