Standardizing analysis of circulating microRNA: clinical and biological relevance.
Author(s): Farina NH, Wood ME, Perrapato SD, Francklyn CS, Stein GS, Stein JL, Lian JB
Publication: J Cell Biochem, 2014, Vol. 115, Page 805-11
PubMed ID: 24357537 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine the effect of prostate cancer diagnosis, freeze-thaw cycling, thaw temperature/duration and microRNA (miRNA, miR) extraction kit on the quantification of miR21-5p, miR-93-5p miR-451a, miR-223-3p, and miR-23a-3p in serum.
Conclusion of Paper
miR-21-5p and miR-93-5p levels were higher and more variable in the serum of patients with prostate cancer than in healthy controls. Specimens thawed twice on ice (for 1-2h) had slightly higher levels of miR-21-5p and miR-23a-3p compared to controls that were thawed once. When specimens were thawed for a second time at 37°C for 30-60 sec, levels of miR-93-5p and miR-451a increased further.
Studies
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Study Purpose
The purpose of this study was to determine the effect of prostate cancer diagnosis, freeze-thaw cycling, and thaw temperature/duration on the quantification of miR21-5p, miR-93-5p miR-451a, miR-223-3p, and miR-23a-3p in serum. Serum obtained from 11 healthy men and 10 men with prostate cancer was aliquoted and stored at -80°C. Serum was thawed on ice for 1-2 h, and aliquoted for immediate extraction or for repeat freezing at -80°C and thawing on ice (slow) or at 37°C (fast) before RNA extraction. RNA was extracted using modifications to the mirVana PARIS or miRNeasy Serum/Plasma kits. The freeze-thaw experiments only included the serum from 6 healthy men and statistical significance was not presented.
Summary of Findings:
miR-21-5p and miR-93-5p levels were higher and more variable in serum from patients with prostate cancer than from healthy controls (p<0.01 and p<0.001, respectively). Compared to specimens thawed once, specimens thawed twice on ice (1-2h each time) had slightly higher levels of miR-21-5p and miR-23a-3p. When specimens were thawed for a second time at 37°C for 30-60 sec, levels of miR-93-5p and miR-451a were higher than in specimens subjected to 1 freeze-thaw cycle.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Diagnosis/ patient condition Healthy
Prostate cancer
Storage Freeze/thaw cycling 1 cycle
2 cycles
Real-time qRT-PCR Specific Targeted nucleic acid miR-21-5p
miR-23a-3p
miR93-5p
miR-451a
Storage Thaw duration 1-2 h
30-60 s
Storage Thaw temperature/condition On ice
37°C