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Evaluating the Effects of Storage Conditions on Multiple Cell-Free RNAs in Plasma by High-Throughput Sequencing.

Author(s): Sun J, Yang X, Wang T, Xing Y, Chen H, Zhu S, Zeng J, Zhou Q, Chen F, Zhang X, Wang WJ

Publication: Biopreserv Biobank, 2022, Vol. , Page

PubMed ID: 36006659 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare the transcriptome and fragmentation of cfRNA (cf-mRNA, cf-lncRNA, cf-miRNA and cf-piRNA) in plasma obtained after a centrifugation delay with plasma that was separated immediately. The changes in expression that were observed in plasma after delayed centrifugation were compared with leukocyte expression.  Blood was collected from two healthy men and two healthy women into EDTA tubes. Blood was stored at room temperature or 4°C for 0, 2, 6 and 24 h before plasma separation by centrifugation at 4°C at 1,600 g for 10 min followed by 12,000 g for 10 min. RNA was extracted from plasma using TRIZOL LS with glycogen as a carrier. Sequencing libraries were constructed using Poly Adenylation Ligation Mediated-Seq and single-end sequenced using a BGISEQ-500RS.  RNA was extracted from the leukocyte pellet and sequenced using the RNAse H method (no further details provided). Differentially expressed genes were identified using DESeq2 using a threshold of ≥2-fold change and an adjusted P-value <0.05. Pathway enrichment was analyzed using Metscape with significance set as a q-value <0.01

   Summary of Findings:

   Principal component analysis (PCA) distinguished plasma specimens stored for 24 h at room temperature before centrifugation from those stored for shorter durations based on the cf miRNA, cf-mRNA, cf-lncRNA and cf-piRNA transcriptome. In contrast, separate clustering of specimens stored for 24 h at 4°C was only observed for cf-mRNA and cf-lncRNAs transcriptomes. Further analysis of cf-mRNAs and cf-lncRNAs in plasma revealed that the majority of fragments were <50 nt in length in specimens that were centrifuged immediately, but a high percentage of cf-mRNA and cf-lncRNA fragments that were 96 nt was observed in specimens processed after a 24 h delay to centrifugation at room temperature and, to a lesser degree, at 4°C.  A centrifugation delay did not alter the fragment size of cf-miRNA and cf-piRNA. Importantly, Pearson’s correlation coefficients decreased and the number of differentially expressed genes increased with longer delays to centrifugation at both temperatures; but, the magnitude of these effects were partially attenuated when plasma was stored at 4°C during the delay. Fewer than 5 cf-mRNAs and cf-lncRNAs were differentially expressed between promptly centrifuged specimens and those stored at room temperature for ≤ 2h or at 4°C ≤ 6 h.  Comparatively, 10 cf-miRNAs and 22 cf-piRNAs were differentially expressed between promptly centrifuged specimens and specimens stored at room temperature for 6 h or at 4°C for 24 h.  A total of 94 cf-tRNAs were differently expressed between specimens stored for 0 and 6 h at room temperature before centrifugation.  mRNA that was elevated in specimens stored at room temperature for 24 h relative to those centrifuged immediately are involved in “myeloid leukocyte activation, hemostasis, regulation of cytokine production, and positive regulation of cell death” and those with lower expression are “involved in the cytoplasmic ribosomal protein complex”. The genes differentially expressed after a 24 h room temperature delay to centrifugation included 8 platelet markers that increased and 4 housekeeping mRNAs (GUSB, ACTB, RAB7A, and HSP90AA1).  As expected, analysis of fragment ends in immediately centrifuged plasma treated with RNase A determined almost 100% of 3’ fragment ends were T or C nucleotides; however, specimens stored for 24 h at room temperature before centrifugation had a significantly lower percentage of fragments ending in either T or C and more introns, indicating they originated from immature RNA and were not RNaseA-cleaved. The correlation coefficient between leukocyte RNA and cfRNA increased progressively with delays to centrifugation. Deconvolution showed that the percentage of cf-mRNA originating from neutrophils increased from 14.3% in immediately centrifuged specimens to 61.2% in specimens centrifuged after 24 h at room temperature (P=0.004).  There were 329 mRNAs and 33 lncRNAs with higher levels in plasma and leukocytes obtained after a 24 h delay at room temperature relative to those obtained immediately. Pathway analysis of these mRNAs showed enrichment of “apoptosis-related pathways, apoptotic and endoplasmic reticulum-nucleus signaling pathways”. There were 231 mRNAs with higher levels in plasma and lower levels in leukocytes after a 24 h room temperature delay to centrifugation; as expected, these mRNAs corresponded to enriched “leukocyte- and immune-related pathways”. A total of 15 tissue enriched genes (TEGs) were identified in plasma but not leukocytes; these included 12 that were liver-specific, and one each that were pancreas-, testis- and intestine-specific.  The expression of TEGs and their fragment size decreased with longer delays to centrifugation.

   Biospecimens

   • Bodily Fluid - Blood
   • Cell - White Cells
   • Bodily Fluid - Plasma

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated
   Biospecimen Aliquots and Components	Blood and blood products	Leukocyte Plasma
   Storage	Storage duration	0 h 2 h 6 h 24 h
   Storage	Storage temperature	Room temperature 4°C

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