Reduction of Systematic Bias in Transcriptome Data from Human Peripheral Blood Mononuclear Cells for Transportation and Biobanking

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.

Genome-wide identification of inter-individually variable DNA methylation sites improves the efficacy of epigenetic association studies

“…Library quality was assessed as previously described. 65 For cluster generation with the HiSeq SR Cluster Kit v4 cBot (Illumina), six libraries were mixed in equimolar concentrations and were loaded into flow cells. Sequencing was performed in the HiSeq 2500 system (Illumina) with the HiSeq SBS Kit v4 (single-end 125-bp reads).…”

Impact of RNA integrity and blood sample storage conditions on the gene expression analysis

“…For example, Ohmomo et al found that after storage at −80°C for a period of time, the quality of the RNA extracted from the peripheral blood mononuclear cells (PBMCs) with RNA protective agent was significantly higher than from the PBMCs without RNA protective agent. 11 However, these methods of blood storage are limited by some objective conditions such as storage equipment, reagents, sample collection and the professionalism of interim management personnel. They may be difficult for onerous clinical procedures of for some more remote transporting after sampling.…”

Intraindividual dynamics of transcriptome and genome-wide stability of DNA methylation

“…We prepared cDNA libraries from 150 ng of total RNA with TruSeq RNA sample Preparation Kit v2 (Illumina, San Diego, CA, USA) for the RNA-Seq, and then carried out fragment size and expression analyses as previously described 22 . For RNA-Seq, equimolar mixtures of the twelve libraries were loaded into four flow cells for cluster generation using a TruSeq Rapid SR Cluster Kit-HS (Illumina) and sequenced on a HiSeq2500 system (Illumina) using a TruSeq Rapid SBS Kit-HS (Illumina), generating 1 × 101 bp reads.…”