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Detection of mRNA in blood samples is emerging as a potential biomarker of diagnosis or prognosis of disease [1, 2]. However, pre-analytical variations in mRNA expression can occur because of a delay in blood processing [3, 4]. In microarray-based transcriptome analysis, gene expression in blood cells was significantly altered by delayed blood sample processing (room temperature ≥ 2 hours) [3, 4]. There are no published studies of the effects of delayed blood sample processing on next generation sequencing for transcriptome analysis. This study was designed to address this.. Blood was collected from a healthy volunteer using 3 EDTA coated tubes. The process of RNA extraction began directly following blood collection, or after cold storage (4°C) for either 24 hours or 48 hours. Using a QIAamp RNA Blood Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions, RNA was extracted and its concentration was measured using a Quant-iTTM RiboGreen RNA Reagent (Invitrogen, Carlsbad, CA, USA). To calculate the RNA Integrity Number (RIN), the RNA 6000 Pico kitwas used and run on an Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Palo Alto, CA, USA). The RNA was then sequenced on the GS Junior platform (Roche, Branford, CT, USA), according to the manufacturer’s instructions. Briefly, double-stranded cDNA was synthesized using the cDNA Synthesis System (Roche Diagnostics, Mannheim, Germany), was fractionated into 600-900 bp fragments via nebulization by compressed nitrogen gas, and then cDNA fragments were ligated to 2 adaptors for 3’ and 5’ ends. Following purification and quantitation, DNA libraries were used for emulsion PCR (emPCR) and bead enrichment. Sequencing was carried out using the GS Junior Titanium Sequencing Kit (Roche Diagnostics). Contig assembly was performed using CLC Genomic Workbench 6 (CLC bio, Aarhus, Denmark). To evaluate the effects of delayed blood processing on RNAseq analysis, concentrations of RNA, RIN, and the number of contigs were assessed from the same volume of blood prepared directly following collection, or after cold storage (4°C) for either 24 hours or 48 hours (Table 1). The concentration of RNA was much lower in blood sample (13 ng/μL) that was stored at 4°C for 48 hours compared to the blood sample (34 ng/μL) that was prepared immediately.

Impact of Time Delay in Processing Blood Sample on Next Generation Sequencing for Transcriptome Analysis