Profiling microRNAs in uncomplicated pregnancies: Serum vs. plasma.
Author(s): Parker VL, Gavriil E, Marshall B, Pacey A, Heath PR
Publication: Biomed Rep, 2021, Vol. 14, Page 24
PubMed ID: 33408858 PubMed Review Paper? No
Purpose of Paper
This paper compared microRNA (miRNA, miR) yield, miRNA percentage, and individual miRNA levels in matched plasma and serum specimens obtained during pregnancy and post-partum. RNA extraction and reverse transcription efficiency as well as the incidence of hemolysis were also compared between plasma and serum specimens.
Conclusion of Paper
The average concentration and percentage miRNA as determined by bioanalyzer were higher in serum than plasma but levels of the four individual miRNAs were generally comparable between plasma and serum at each pregnancy timepoint. However, plasma obtained six weeks postpartum had higher miR-451 and miR-30e-5p than matched serum. The standard deviation of the quantification cycle values (Cq) of each of the miRNA were also comparable in plasma and serum at most timepoints. There was no difference in RNA extraction or reverse transcription efficiency or incidence of hemolysis between serum and plasma.
Studies
-
Study Purpose
This study compared miRNA yield, miRNA percentage, and individual miRNA levels in matched plasma and serum specimens obtained during pregnancy and post-partum. RNA extraction and reverse transcription efficiency as well as the incidence of hemolysis were also compared between plasma and serum specimens. Blood was collected using 21-gauge needles into three sodium citrate Vacutainer tubes and one SST Vacutainer from each of two women during the second trimester of pregnancy, three women during weeks 36-40 of pregnancy and five women 6 weeks postpartum. Tubes were stored upright on ice for 4-6 h before centrifugation at 1900 x g for 10 min. The supernatants (plasma and serum) were stored at -80°C. Plasma was thawed and recentrifuged at 16,000 x g for 10 min at 4˚C to eliminate platelets. UniSp2, UniSp4, and UniSp6 were added and RNA was extracted from plasma and serum using the Maxwell® Rapid Sample Concentrator (RSC) miRNA Tissue or Plasma Serum Kit and stored at -80°C. RNA and miRNA yield and miRNA percentage were evaluated using an Agilent Small RNA Chip and 2100 Bioanalyzer. miRNA was reverse-transcribed using the qScript microRNA cDNA Synthesis Kit and levels of UniSp2 (extraction control), cel‑miR‑39‑3p (reverse transcription control), miR‑222‑3p, miR‑23a, miR‑30e‑5p, and miR‑451a were quantified by real-time PCR and levels were normalized to the geomean of let‑7i‑3p and miR‑148‑3p.
Summary of Findings:
The average concentration and percentage miRNA as determined by bioanalyzer were higher in serum than plasma (P=0.0039 and P=0.0008, respectively) but levels of individual miRNAs were generally comparable between plasma and serum at all timepoints. However, plasma obtained six weeks postpartum had higher miR-451 and miR-30e-5p levels than matched serum (P=0.03 and P=0.008, respectively). The standard deviation of the Cq was also generally comparable in plasma and serum except for miR-30e-5p which showed more variability in plasma than serum obtained during weeks 36-40 of pregnancy. Further, there was no difference in RNA extraction or reverse transcription efficiency (UniSp2 and cel-miR-39-3p) between serum and plasma and hemolysis was not observed in any of the specimens (difference in mean Cq of mir-23 and miR-451 ≤7 cycles).
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Pregnant
Platform:
Analyte Technology Platform RNA Automated electrophoresis/Bioanalyzer RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Real-time qRT-PCR Specific Targeted nucleic acid miR-451
miR-30e-5p
miR 222 3p
miR-23a
Biospecimen Aliquots and Components Blood and blood products Plasma
Serum