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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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Workflow of the study design.
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pone-0104283-g001: Workflow of the study design.

Mentions: The workflow for this study is shown in Figure 1. Forty milliliters of whole blood was collected from 11 healthy volunteers (mean age, 35.3±10.6 years; eight men and three women) into five BD Vacutainer CPT tubes with sodium heparin (8 mL; Becton Dickinson and Company) through a 21-gauge needle. PBMCs were separated by centrifugation (Sorvall Legend XFR; Thermo Fisher Scientific, Waltham, MA) at 1,700×g for 20 min at room temperature. To remove any contaminating platelets and plasma, PBMCs were washed three times in 30 mL phosphate buffer saline (PBS) containing 2 mM EDTA, followed by centrifugation at 250×g for 10 min at room temperature [18]. PBMCs were then resuspended in 1 mL PBS, and cell numbers were determined using a C-Chip disposable hemocytometer (Biochrom AG, Berlin, Germany).


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)

Workflow of the study design.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0104283-g001: Workflow of the study design.
Mentions: The workflow for this study is shown in Figure 1. Forty milliliters of whole blood was collected from 11 healthy volunteers (mean age, 35.3±10.6 years; eight men and three women) into five BD Vacutainer CPT tubes with sodium heparin (8 mL; Becton Dickinson and Company) through a 21-gauge needle. PBMCs were separated by centrifugation (Sorvall Legend XFR; Thermo Fisher Scientific, Waltham, MA) at 1,700×g for 20 min at room temperature. To remove any contaminating platelets and plasma, PBMCs were washed three times in 30 mL phosphate buffer saline (PBS) containing 2 mM EDTA, followed by centrifugation at 250×g for 10 min at room temperature [18]. PBMCs were then resuspended in 1 mL PBS, and cell numbers were determined using a C-Chip disposable hemocytometer (Biochrom AG, Berlin, Germany).

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