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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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Protocol to reduce systematic bias for transcriptome analysis of PBMCs collected in remote assessment centers.A protocol for pre-analytical operations to mediate the effects of systematic bias in transcriptome data of PBMCs for transportation and biobanking is shown.
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pone-0104283-g005: Protocol to reduce systematic bias for transcriptome analysis of PBMCs collected in remote assessment centers.A protocol for pre-analytical operations to mediate the effects of systematic bias in transcriptome data of PBMCs for transportation and biobanking is shown.

Mentions: Protocols for sample collection for large-scale biobanks must be as simple and as low cost as possible. Based on the results of the present study, we established a protocol for pre-analytical treatment of samples at remote assessment centers to obtain high-quality RNA from PBMCs at a central laboratory (Figure 5). For blood collection (Procedure 1), we used a Vacutainer CPT tube, which could help to reduce contamination of red blood cells in the PBMC layer without the need for technical expertise. After centrifugation (Procedure 2), extracted PBMCs were washed with more than 15 volumes of 10 mM PBS containing 2 mM EDTA (pH 7.2) to remove platelets (Procedures 3–6). Then, PBMCs are resuspended in a suitable volume of 10 mM PBS (Procedures 7–9). We assumed that RNA extraction was performed using a Maxwell16 instrument at a central laboratory (Procedure 15). Considering the cost of reagents, we selected 1-Thio, which is supplied with the Maxwell16 LEV simplyRNA Blood Kit, as a stabilizing reagent (Procedure 10). After this pretreatment, the tubes were frozen at −80°C for at least 24 h and at most one week (Procedure 11). For these pre-analytical operations, a cool incubator, a deep freezer, a tabletop centrifuge, a tabletop clean bench, and an aspirator are required at remote assessment centers. This equipment can be arranged within an area of 2×2 m.


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)

Protocol to reduce systematic bias for transcriptome analysis of PBMCs collected in remote assessment centers.A protocol for pre-analytical operations to mediate the effects of systematic bias in transcriptome data of PBMCs for transportation and biobanking is shown.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0104283-g005: Protocol to reduce systematic bias for transcriptome analysis of PBMCs collected in remote assessment centers.A protocol for pre-analytical operations to mediate the effects of systematic bias in transcriptome data of PBMCs for transportation and biobanking is shown.
Mentions: Protocols for sample collection for large-scale biobanks must be as simple and as low cost as possible. Based on the results of the present study, we established a protocol for pre-analytical treatment of samples at remote assessment centers to obtain high-quality RNA from PBMCs at a central laboratory (Figure 5). For blood collection (Procedure 1), we used a Vacutainer CPT tube, which could help to reduce contamination of red blood cells in the PBMC layer without the need for technical expertise. After centrifugation (Procedure 2), extracted PBMCs were washed with more than 15 volumes of 10 mM PBS containing 2 mM EDTA (pH 7.2) to remove platelets (Procedures 3–6). Then, PBMCs are resuspended in a suitable volume of 10 mM PBS (Procedures 7–9). We assumed that RNA extraction was performed using a Maxwell16 instrument at a central laboratory (Procedure 15). Considering the cost of reagents, we selected 1-Thio, which is supplied with the Maxwell16 LEV simplyRNA Blood Kit, as a stabilizing reagent (Procedure 10). After this pretreatment, the tubes were frozen at −80°C for at least 24 h and at most one week (Procedure 11). For these pre-analytical operations, a cool incubator, a deep freezer, a tabletop centrifuge, a tabletop clean bench, and an aspirator are required at remote assessment centers. This equipment can be arranged within an area of 2×2 m.

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