Quantification of Circulating miRNAs in Plasma: Effect of Preanalytical and Analytical Parameters on Their Isolation and Stability.
Author(s): Sourvinou IS, Markou A, Lianidou ES
Publication: J Mol Diagn, 2013, Vol. 15, Page 827-34
PubMed ID: 23988620 PubMed Review Paper? No
Purpose of Paper
Conclusion of Paper
Studies
-
Study Purpose
The purpose of this study was to determine the effects of plasma volume, RNA precipitation temperature during TriZol LS extraction, and miRNA extraction kit type on the recovery of spiked cel-miR-39 and endogenous hsa-miR-21 in EDTA plasma from healthy individuals and patients with non-small cell lung carcinomas. EDTA blood was centrifuged and the was plasma spiked with cel-miR-39 before miRNA extraction.
Summary of Findings:
Using the TriZol LS kit, the highest recovery of cel-miR-39 and hsa-miR-21 from plasma was achieved when the least volume of plasma was used (200 uL) and when RNA was precipitated at room temperature for 10 min rather than at -20 degrees C overnight. The yields of cel-miR-39 and hsa-miR-21 were highest using the mirVana PARIS kit for extraction and lowest using TRIzol LS (with optimized protocol) or the Norgen miRNA purification kit.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Normal
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte isolation method TRIzol LS
miRNeasy mini kit
mirVana PARIS kit
Norgen miRNA purification kit
Analyte Extraction and Purification Analyte purification Precipitation at room temperature
Precipitation at -20 degrees C
Biospecimen Aliquots and Components Aliquot size/volume 200 uL
250 uL
300 uL
500 uL
Real-time qRT-PCR Specific Targeted nucleic acid hsa-miR-21
cel-miR-39
-
Study Purpose
The purpose of this study was to determine the effects of storage temperature and duration for plasma or extracted miRNA on levels of cel-miR-39, hsa-miR-21 and hsa-miR-16 in EDTA plasma from healthy individuals and patients with non-small cell lung carcinomas. EDTA blood was centrifuged and plasma was spiked with cel-miR-39 before miRNA extraction. Plasma was stored for up to 4 months, while extracted cel-miR-39 was stored for up to 1 year.
Summary of Findings:
CT values for hsa-miR-21 and hsa-miR-16 increased with storage of plasma at 4, -20 or -70 degrees C, and significant increases, compared to 24 h of storage, were observed after 1 and 4 months at all three storage temperatures (p<0.05, all). However, the CT values of hsa-miR-21 and hsa-miR-16 increased by 3-4 cycles after storage of plasma at -20 or -70 degrees C, while the CT values of hsa-miR-21 and hsa-miR-16 increased by 14-15 cycles after storage of plasma at 4 degrees C for 4 months. Consequently, the authors conclude storage of plasma at -20 or -70 degrees C is better than storage of plasma at 4 degrees C. After extraction, cel-miR-39 was stable at -70 degrees C for at least 1 year.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Neoplastic - Carcinoma
- Normal
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 24 h
48 h
1 month
2 months
4 months
6 months
8 months
9 months
12 months
Storage Storage temperature -70 degrees C
-20 degrees C
4 degrees C
Real-time qRT-PCR Specific Targeted nucleic acid hsa-miR-21
hsa-miR-16