Factors influencing degradation kinetics of mRNAs and half-lives of microRNAs, circRNAs, lncRNAs in blood in vitro using quantitative PCR.
Author(s): Wang C, Liu H
Publication: Sci Rep, 2022, Vol. 12, Page 7259
PubMed ID: 35508612 PubMed Review Paper? No
Purpose of Paper
This paper investigated potential effects of storage of blood and/or RNA at room temperature, and storage of complementary DNA (cDNA) at -20°C on β-actin levels; the calculated half-life of total RNA, messenger RNA (mRNA), microRNA (miRNA, miR), long non-coding RNA (lncRNA), and circular RNA (circRNA) was also investigated in blood specimens stored at room temperature.
Conclusion of Paper
Quantification cycle (Cq) values for β-actin were lowest when RNA was extracted, reverse transcribed and used for real-time PCR without delay and progressively increased when blood or RNA was stored at room temperature, or when cDNA was stored at -20°C. The largest effects were attributed to storage of blood followed by storage of cDNA. The half-life of total RNA (quantified spectrophotometrically) in blood stored at room temperature was calculated to be 14.4 h. The half-life of mRNA and miRNA in blood stored at room temperature were each 16.4 h, while the half-life of circRNA and lncRNA were 24.56 and 17.46 h, respectively.
Studies
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Study Purpose
This study investigated potential effects of storage of blood and/or RNA at room temperature, and storage of cDNA at -20°C on β-actin levels; the calculated half-life of total RNA, mRNA, miRNA, lncRNA, and circRNA was also investigated in blood specimens stored at room temperature. Aliquots of K2EDTA blood were collected from an unspecified number of people (no other collection details were provided). Genomic RNA was extracted using a blood RNA extraction kit from TIANGEN and stored for 0, 12 or 24 h before reverse transcription. RNA was reverse transcribed using an unspecified method. cDNA was stored for 0, 12 or 24 h at -20°C before quantification of β-actin by real-time PCR. To investigate potential effects of storing blood and/or extracted RNA, each were stored at room temperature for 0, 12, or 24 h. To calculate the half-life of RNA in blood, RNA was extracted from blood every 12 h and RNA concentration was quantified by spectrophotometer (total RNA) and by real-time PCR amplification of mRNA (GAPDH and β-actin), miRNA (miR-221, miR-16-1, miR-126, miR-145, and miR-28-3p, respectively), lncRNA (LncRNA GASL, LncRNA GASL, STEAP3-AS1, NR-038263 and LncRNASNHG5, respectively) and circRNA (Circ002532, circ_0000190, circ_0001785, circ_0000520 and circARIDIB).
Summary of Findings:
β-actin Cq values increased linearly with the natural log of the dilution factor (R=0.9759-0.9997). β-actin Cq values were lowest when RNA was extracted, reverse transcribed and used for real-time PCR without delay. Storage of blood or extracted RNA at room temperature or cDNA at -20°C led to progressively increased β-actin Cq values. Multivariate analysis found significant effects of blood storage at room temperature, RNA storage at room temperature, and cDNA storage at -20°C on β-actin Cq values (P<0.001, P=0.037, and P<0.001, respectively), with the largest effects attributed to storage of blood followed by storage of cDNA. The half-life of total RNA (quantified spectrophotometrically) in blood stored at room temperature was calculated to be 14.4 h. The half-life of mRNA and miRNA in blood stored at room temperature were each 16.4 h, but the half-life of circRNA and lncRNA were 24.56 and 17.46 h, respectively.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR RNA Spectrophotometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 12 h
24 h
0 h
Storage Storage conditions As blood at room temperature
As RNA at room temperature
As cDNA at -20°C
Real-time qRT-PCR Specific Targeted nucleic acid mRNA
miRNA
lncRNA
circRNA
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated