Intraindividual Temporal miRNA Variability in Serum, Plasma, and White Blood Cell Subpopulations.
Author(s): Ammerlaan W, Betsou F
Publication: Biopreserv Biobank, 2016, Vol. 14, Page 390-397
PubMed ID: 27096687 PubMed Review Paper? No
Purpose of Paper
This paper evaluated the variability in microRNA (miRNA, miR) levels in serum, plasma, and white blood cell (WBC) populations among specimens collected from two healthy individuals over the course of one year.
Conclusion of Paper
Hemoglobin levels were <1.4 mg/mL in all specimens. Yields of sorted CD45+, CD3+, CD14+, and CD15+ cells were reproducible over the course of the year and RNA isolated from all WBC subpopulations had RNA integrity numbers (RIN)f ≥7. While a small percentage of miRNA were classified as extremely stable over the collection period (<10% CV), 3.9-7.8% of miRNAs were classified as very unstable (CV 110-140%) and 9-19.6% of miRNAs were classified as extremely unstable (CV >140%). The authors concluded that the temporal intraindividual variability of miRNAs must be considered during biomarker validation.
Studies
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Study Purpose
This study evaluated the variability in miRNA levels in serum, plasma, and WBC populations among specimens collected from two healthy individuals over the course of one year. Blood was collected into EDTA Vacutainer and CAT Vacutainer tubes every 2-3 months from two healthy volunteers (a 62 year-old male and a 46-year-old female) between 9 and 11:00 AM. Within 2 h of collection, EDTA plasma was separated by centrifugation at 2,000 g for 20 min and aliquots were stored at -80°C. Serum was separated from clotted blood in CAT tubes by centrifugation at 2,000 g for 10 min and stored at -80°C. Blood counts were enumerated using the ABX Micros CRP 200 machine and WBC subpopulations were quantified using an autoMACS Pro Separator machine. Subpopulations were separated by flow cytometry using antibodies against CD3, CD14, CD15, and CD45. Hemoglobin levels were quantified using a Synergy MX spectrophotometer. Thawed plasma and serum were centrifuged at 3,000 g for 5 min at 4°C before RNA isolation. RNA was isolated from plasma, serum and isolated cells using QIAzol and purified with RNeasy Mini spin columns. RNA was stored at -80°C. RNA was quantified by spectrophotometer and integrity was analyzed using the RNA Nano Chip on a Bioanalyzer 2100. RNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit and miRNAs were quantified by real-time PCR using the SmartChip Human miRNA Panel v3.0.
Summary of Findings:
Hemoglobin levels were <1.4 mg/mL in all specimens. Yields of sorted CD45+, CD3+, CD14+, and CD15+ cells were reproducible over the course of the year. RNA isolated from all WBC subpopulations had RNA integrity numbers (RIN) of ≥7. While a small percentage (0.7-4.6%) of miRNA were classified as extremely stable during the specimen collection period (<10% CV), 3.9-7.8% of miRNAs were classified as very unstable (CV 110-140%) and 9-19.6% of miRNAs were classified as extremely unstable (CV >140%). The most variable miRNA in plasma was miR-1260 and the most variable miRNAs in serum were miR-101*, miR-146B-3P, and miR-218-1*. Among the most stable miRNAs shard by both serum and plasma were miR-106B*, miR-3652, miR-503, and miR-99A (CV <30%). The most stable miRNA shared among the WBC subpopulations were miR-4289, miR-4297, miR-3158, and LET-7A-2* (CV<20%). The authors concluded that the temporal intraindividual variability of miRNAs must be considered during biomarker validation.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR RNA Automated electrophoresis/Bioanalyzer Cell count/volume Flow cytometry Protein Spectrophotometry RNA Spectrophotometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Time of biospecimen collection Multiple specimens collected over the course of 1 year
Biospecimen Aliquots and Components Blood and blood products Serum
Plasma
White blood cells