MicroRNA Isolation by Trizol-Based Method and Its Stability in Stored Serum and cDNA Derivatives.
Author(s): Trakunram K, Champoochana N, Chaniad P, Thongsuksai P, Raungrut P
Publication: Asian Pac J Cancer Prev, 2019, Vol. 20, Page 1641-1647
PubMed ID: 31244282 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of extraction method used, serum volume, storage of serum and extracted microRNA (miRNA, miR), and freeze/thaw cycling of extracted miRNA on the levels of miR-20a, miR-145, and RNU6 in serum.
Conclusion of Paper
The RNA yield was lower when extracted using Trizol LS rather than miRNeasy but the purity (A260/280 ad A260/230) was comparable between the two methods. RNA concentration increased with increasing serum volume. Storage of serum or extracted miRNA at room temperature resulted in significant increases in the cycle threshold (CT) values for miR-20a, miR-145, and RNU6 but the magnitude of increase and significance depended on the miRNA in question. However, there was no effect of storing serum or extracted miRNA at 4°C, -20°C, or -80°C and up to four freeze/thaw cycles on CT values for miR-20a, miR-145, or RNU6.
Studies
-
Study Purpose
This study investigated the effects of extraction method used, serum volume, storage of serum and extracted miRNA, and freeze/thaw cycling of extracted miRNA on the levels of miR-20a, miR-145, and RNU6 in serum. Blood was collected from healthy volunteers into Greiner Bio-One clotting tubes. After 30 min clotting at room temperature, serum was isolated by centrifugation at 3400 x g for 10 min followed by filtration through a 0.22 µm filter. Serum was aliquoted and stored at room temperature, 4°C, -20°C, or -80°C for 0, 1, 3, 5, 24, 48, or 72 h before being frozen at an unspecified temperature until RNA isolation. RNA was isolated from plasma using the miRNeasy Serum/Plasma Kit or Trizol LS. RNA concentration and purity was determined spectrophotometrically. Extracted RNA was stored for 0 or 3 months at -20°C before quantification of miR-145, miR-20a, and RNU6 by real-time PCR using the miScript SYBR Green PCR kit. The effect of freeze/thaw cycling of extracted miRNAs was investigated by thawing at 4°C for 1 h one to four times before analysis.
Summary of Findings:
The RNA yield was 35% lower when extracted using Trizol LS rather than miRNeasy but the purity (A260/280 ad A260/230) was comparable between the two methods. RNA concentration increased with increasing serum volume from 9.75 ng/µL when 50 µL was used to 25.96 ng/µL when 200 µL was used. Storage of serum at room temperature resulted in a significant increase in the CT value for miR-20a and miR-145 after only 1 h (P<0.05, both) and RNU6 after 24 h (-15%, P=0.008) but no changes were observed in CT values for miR-20a, miR-145, or RNU6 when serum was stored for up to 72 h at 4°C, -20°C, or -80°C. Similarly, there was no effect of storing extracted miRNA at 4°C, -20°C, or -80°C on CT values for miR-20a, miR-145, or RNU6 but storage at room temperature resulted in higher CT values for miR-20a, miR-145, and RNU6 (P<0.05, all). Further, there was no effect of up to four freeze/thaw cycles on the CT values for miR-20a, miR-145, or RNU6.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR RNA Spectrophotometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Aliquot size/volume 50 µL
100 µL
150 µL
200 µL
Analyte Extraction and Purification Analyte isolation method Trizol LS reagent
miRNeasy mini kit
Storage Storage temperature Room temperature
4°C
-20°C
-80°C
Real-time qRT-PCR Specific Targeted nucleic acid miR-20a
miR-145
RNU6
Storage Storage conditions As serum
As extracted miRNA
Storage Freeze/thaw cycling 0 cycles
1 cycle
2 cycles
3 cycles
4 cycles
Storage Storage duration 0 h
1 h
3 h
5 h
24 h
48 h
72 h