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Reduction of systematic bias in transcriptome data from human peripheral blood mononuclear cells for transportation and biobanking.

Ohmomo H, Hachiya T, Shiwa Y, Furukawa R, Ono K, Ito S, Ishida Y, Satoh M, Hitomi J, Sobue K, Shimizu A - PLoS ONE (2014)

Bottom Line: RNA-Seq for 25,223 transcripts also suggested that about 40% of transcripts were systematically biased.On the basis of the results of this study, we established a protocol to reduce systematic bias in the expression levels of RNA transcripts isolated from PBMCs.We believe that these data provide a novel methodology for collection of high-quality RNA from PBMCs for biobank researchers.

View Article: PubMed Central - PubMed

Affiliation: Division of Biobank and Data Management, Iwate Tohoku Medical Megabank Organization, Iwate Medical University, Shiwa-gun, Iwate, Japan.

ABSTRACT
Transportation of samples is essential for large-scale biobank projects. However, RNA degradation during pre-analytical operations prior to transportation can cause systematic bias in transcriptome data, which may prevent subsequent biomarker identification. Therefore, to collect high-quality biobank samples for expression analysis, specimens must be transported under stable conditions. In this study, we examined the effectiveness of RNA-stabilizing reagents to prevent RNA degradation during pre-analytical operations with an emphasis on RNA from peripheral blood mononuclear cells (PBMCs) to establish a protocol for reducing systematic bias. To this end, we obtained PBMCs from 11 healthy volunteers and analyzed the purity, yield, and integrity of extracted RNA after performing pre-analytical operations for freezing PBMCs at -80°C. We randomly chose 7 samples from 11 samples individually, and systematic bias in expression levels was examined by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), RNA sequencing (RNA-Seq) experiments and data analysis. Our data demonstrated that omission of stabilizing reagents significantly lowered RNA integrity, suggesting substantial degradation of RNA molecules due to pre-analytical freezing. qRT-PCR experiments for 19 selected transcripts revealed systematic bias in the expression levels of five transcripts. RNA-Seq for 25,223 transcripts also suggested that about 40% of transcripts were systematically biased. These results indicated that appropriate reduction in systematic bias is essential in protocols for collection of RNA from PBMCs for large-scale biobank projects. Among the seven commercially available stabilizing reagents examined in this study, qRT-PCR and RNA-Seq experiments consistently suggested that RNALock, RNA/DNA Stabilization Reagent for Blood and Bone Marrow, and 1-Thioglycerol/Homogenization solution could reduce systematic bias. On the basis of the results of this study, we established a protocol to reduce systematic bias in the expression levels of RNA transcripts isolated from PBMCs. We believe that these data provide a novel methodology for collection of high-quality RNA from PBMCs for biobank researchers.

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Related in: MedlinePlus

qRT-PCR analysis of 19 target transcripts.GAPDH and the Ctrl1 condition were used as a reference transcript and condition, respectively. ΔΔCt values are shown by vertical axes. Horizontal axes represent pre-analytical conditions in the following order: Ctrl2, Without stab, Protect, Lock, Stab, SDS, and 1-Thio. *, p<0.05; **, p<0.01 (Wilcoxon signed rank test).
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pone-0104283-g002: qRT-PCR analysis of 19 target transcripts.GAPDH and the Ctrl1 condition were used as a reference transcript and condition, respectively. ΔΔCt values are shown by vertical axes. Horizontal axes represent pre-analytical conditions in the following order: Ctrl2, Without stab, Protect, Lock, Stab, SDS, and 1-Thio. *, p<0.05; **, p<0.01 (Wilcoxon signed rank test).

Mentions: To examine whether gene expression in PBMCs was biased during pre-analytical operations and whether the bias could be reduced using stabilizing reagents, we performed qRT-PCR assays to analyze the expression of 21 transcripts from seven individuals for each of the eight conditions. When GAPDH was used as control, no significant bias in ΔCt was observed between the Ctrl1 and Ctrl2 conditions for all target transcripts (Figure 2), suggesting that our experimental results were consistent between replicates. Between the Without stab and Ctrl1 conditions, five transcripts (FOS, SPI1, IL6, HSPA1A, and BDNF) were significantly biased (significance level of 0.05), indicating that absence of a stabilizing reagent promoted bias of transcript expression in a significant portion (∼40%) of transcripts. Under the Protect, Lock, Stab, SDS, and 1-Thio conditions, nine, three, one, five, and one transcripts were significantly biased, respectively, compared to the Ctrl1 condition (Figure 2). These results indicated that gene expression bias during pre-analytical operations was reduced under the Lock, Stab, and 1-Thio conditions. Similar results were obtained when ACTB was used as control (Figure S1).


Reduction of systematic bias in transcriptome data from human peripheral blood mononuclear cells for transportation and biobanking.

Ohmomo H, Hachiya T, Shiwa Y, Furukawa R, Ono K, Ito S, Ishida Y, Satoh M, Hitomi J, Sobue K, Shimizu A - PLoS ONE (2014)

qRT-PCR analysis of 19 target transcripts.GAPDH and the Ctrl1 condition were used as a reference transcript and condition, respectively. ΔΔCt values are shown by vertical axes. Horizontal axes represent pre-analytical conditions in the following order: Ctrl2, Without stab, Protect, Lock, Stab, SDS, and 1-Thio. *, p<0.05; **, p<0.01 (Wilcoxon signed rank test).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4125218&req=5

pone-0104283-g002: qRT-PCR analysis of 19 target transcripts.GAPDH and the Ctrl1 condition were used as a reference transcript and condition, respectively. ΔΔCt values are shown by vertical axes. Horizontal axes represent pre-analytical conditions in the following order: Ctrl2, Without stab, Protect, Lock, Stab, SDS, and 1-Thio. *, p<0.05; **, p<0.01 (Wilcoxon signed rank test).
Mentions: To examine whether gene expression in PBMCs was biased during pre-analytical operations and whether the bias could be reduced using stabilizing reagents, we performed qRT-PCR assays to analyze the expression of 21 transcripts from seven individuals for each of the eight conditions. When GAPDH was used as control, no significant bias in ΔCt was observed between the Ctrl1 and Ctrl2 conditions for all target transcripts (Figure 2), suggesting that our experimental results were consistent between replicates. Between the Without stab and Ctrl1 conditions, five transcripts (FOS, SPI1, IL6, HSPA1A, and BDNF) were significantly biased (significance level of 0.05), indicating that absence of a stabilizing reagent promoted bias of transcript expression in a significant portion (∼40%) of transcripts. Under the Protect, Lock, Stab, SDS, and 1-Thio conditions, nine, three, one, five, and one transcripts were significantly biased, respectively, compared to the Ctrl1 condition (Figure 2). These results indicated that gene expression bias during pre-analytical operations was reduced under the Lock, Stab, and 1-Thio conditions. Similar results were obtained when ACTB was used as control (Figure S1).

Bottom Line: RNA-Seq for 25,223 transcripts also suggested that about 40% of transcripts were systematically biased.On the basis of the results of this study, we established a protocol to reduce systematic bias in the expression levels of RNA transcripts isolated from PBMCs.We believe that these data provide a novel methodology for collection of high-quality RNA from PBMCs for biobank researchers.

View Article: PubMed Central - PubMed

Affiliation: Division of Biobank and Data Management, Iwate Tohoku Medical Megabank Organization, Iwate Medical University, Shiwa-gun, Iwate, Japan.

ABSTRACT
Transportation of samples is essential for large-scale biobank projects. However, RNA degradation during pre-analytical operations prior to transportation can cause systematic bias in transcriptome data, which may prevent subsequent biomarker identification. Therefore, to collect high-quality biobank samples for expression analysis, specimens must be transported under stable conditions. In this study, we examined the effectiveness of RNA-stabilizing reagents to prevent RNA degradation during pre-analytical operations with an emphasis on RNA from peripheral blood mononuclear cells (PBMCs) to establish a protocol for reducing systematic bias. To this end, we obtained PBMCs from 11 healthy volunteers and analyzed the purity, yield, and integrity of extracted RNA after performing pre-analytical operations for freezing PBMCs at -80°C. We randomly chose 7 samples from 11 samples individually, and systematic bias in expression levels was examined by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), RNA sequencing (RNA-Seq) experiments and data analysis. Our data demonstrated that omission of stabilizing reagents significantly lowered RNA integrity, suggesting substantial degradation of RNA molecules due to pre-analytical freezing. qRT-PCR experiments for 19 selected transcripts revealed systematic bias in the expression levels of five transcripts. RNA-Seq for 25,223 transcripts also suggested that about 40% of transcripts were systematically biased. These results indicated that appropriate reduction in systematic bias is essential in protocols for collection of RNA from PBMCs for large-scale biobank projects. Among the seven commercially available stabilizing reagents examined in this study, qRT-PCR and RNA-Seq experiments consistently suggested that RNALock, RNA/DNA Stabilization Reagent for Blood and Bone Marrow, and 1-Thioglycerol/Homogenization solution could reduce systematic bias. On the basis of the results of this study, we established a protocol to reduce systematic bias in the expression levels of RNA transcripts isolated from PBMCs. We believe that these data provide a novel methodology for collection of high-quality RNA from PBMCs for biobank researchers.

Show MeSH
Related in: MedlinePlus