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The impact of pre-freezing storage time and temperature on gene expression of blood collected in EDTA tubes.

Author(s): Martire S, Valentino P, Marnetto F, Mirabile L, Capobianco M, Bertolotto A

Publication: Mol Biol Rep, 2022, Vol. , Page

PubMed ID: 35279776 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to evaluate the effects of storing blood collected from healthy individuals at room temperature or 4°C for up to 24 h before isolation of PBMCs on RNA yield, RNA purity and the levels of 4 housekeeping and 8 target RNAs. Potential effects associated with the house-keeping gene used for normalization of target RNA levels were also investigated.  Blood was collected from 10 healthy donors into K₂EDTA tubes. PBMCs were isolated from blood using Lymphoprep after 0, 2, 4, 6 and 24 h at room temperature (21°C) and 4°C. PBMCs were stored at -80°C in RNAlater until RNA extraction with the Maxwell R 16LEV simply RNA Tissue Kit. RNA yield and purity were assessed by spectrophotometer, and RNA integrity was evaluated using the Total RNA Nano Kit on a bioanalyzer.  RNA was reverse transcribed with the High-Capacity cDNA Reverse Transcription Kit. Levels of B2M, CASC3, GAPDH, HPRT1, CD14, CD19, CD20, interleukin 10 (IL10), myxovirus resistance protein 1 (MxA), tumor necrosis factor (TNF), TNF alpha induced protein 3 (TNFAIP3), and nuclear receptor subfamily 4 group A member 2 (NR4A2) were quantified by real-time PCR.  The 24 h timepoint was included in graphs separately and not included in any statistical analysis.

   Summary of Findings:

   While storage of blood at 6 h at either room temperature or 4°C prior to PBMC isolation did not affect RNA yield or purity (A260/280), storage for 24 h at either temperature resulted in lower RNA yields and RNA integrity numbers (RINs) below 5 for some specimens (three with storage at 4°C and one at room temperature). CT values of each of the 4 housekeeping genes investigated increased progressively with a delay before PBMC isolation, although significant changes were limited to those specimens stored at 4°C. Compared to immediately isolated PBMCs, PBMCs isolated from blood stored at 4°C for 2 h had higher CT values CASC3, HPRT and GAPDH CT values, while blood stored at 4°C  for 4 h had higher HPRT and GAPDH CT values, and blood stored at 4°C  for 6 h had higher GAPDH CT values (P<0.05, all).  When levels of the targeted RNA were normalized to β₂ macroglobulin (B2M) fewer significant differences were observed between PBMCs that were isolated immediately and those isolated after blood storage than when levels were normalized to HPRT1.  Levels of B2M-normalized NR4A2 were higher in blood that was stored at room temperature for 4 h or at 4°C for 2 h (P<0.05, both) compared to immediately processed specimens. Levels of B2M-normalized TNFAIP3 were higher in PBMCs isolated from blood stored for 2 or 4 h compared to levels in immediately isolated PBMCs (P<0.05 and P<0.001, respectively). When normalized to HPRT1, significant differences in levels of IL10, MXA, TNF, TNFAIP3, and NR4A2 were observed depending on the temperature and duration of the delay to PBMC isolation.

   Biospecimens

   • Cell - White Cells
   • Bodily Fluid - Blood

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	Real-time qRT-PCR
   RNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage temperature	4°C Room temperature
   Storage	Storage duration	0 h 2 h 4 h 6 h 24 h
   Real-time qRT-PCR Specific	Targeted nucleic acid	B2M CASC3 HPRT1 GAPDH TNF CD14 CD19 CD20 IL10 MxA TNFAIP3 NR4A2
   Real-time qRT-PCR Specific	Data handling	Normalized to B2M Normalized to HPRT1

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