Protocol for miRNA isolation from biofluids.
Author(s): Lekchnov EA, Zaporozhchenko IA, Morozkin ES, Bryzgunova OE, Vlassov VV, Laktionov PP
Publication: Anal Biochem, 2016, Vol. 499, Page 78-84
PubMed ID: 26874020 PubMed Review Paper? No
Purpose of Paper
This paper describes the optimization of a guanidium thiocyanate and octanoic acid (Gu/OcA) method for the isolation of microRNA (miRNA, miR) from plasma and urine and compared the efficiency of miRNA isolation with this method to the miRCURY kit and an acid phenol:/chloroform-based method. The authors further explored the differences between the Gu/OcA and miRCURY kit by investigating protein removal.
Conclusion of Paper
Optimization of the Gu/OcA protocol to obtain the lowest cycle threshold values for miR-16 and miR-126 identified 0.6 M guanidium, 0.5% octanoic acid at pH 4 as the best for plasma and 1.35 M guanidium, 1.5% octanoic acid at pH 4 as the best for urine. The optimized Gu/OcA method was more efficient at extracting miR-16 and miR-126 from plasma and urine than either the miRCURY kit or the acid phenol:chloroform method. As determined by A260/A280 and SDS-PAGE, the miRCURY kit was better at removing proteins from blood than the Gu/OcA method but not as effective at eliminating biopolymers from urine. The authors postulate this more aggressive precipitation of proteins from plasma resulted in loss of miRNAs through co-precipitation.
Studies
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Study Purpose
This study describes the optimization of the guanidium and octanoic acid concentrations and the pH for the isolation of microRNA (miRNA, miR) from plasma and urine and compared the efficiency of miRNA isolation with Gu/OcA with that obtained using the miRCURY kit and an acid phenol:chloroform-based method. The authors further explored the differences between the Gu/OcA and miRCURY kit by investigating protein removal. Venous blood collected in EDTA vacutainers and urine were obtained from 10 healthy individuals. Plasma was processed from blood within 4 h of collection by centrifugation at 290 x g for 20 min followed by centrifugation at 1200 x g for 20 min. Urine was centrifuged at 400 x g for 20 min followed by at 17,000 x g for 20 min. Urine and plasma supernatants were frozen at -20˚C. Before miRNA extraction using each protocol, specimens were thawed, mixed, and centrifuged at 3000 x g for 5 min. To optimize Gu/OcA extraction, a variety of concentrations of guanidium thiocyanate (0.3, 0.6, 0.9, and 1.35 M), octanoic acid (0.5%, and 1.5%) and pH values (4, 5, and 6) were tried in combination. To compare the Gu/OcA method with other methods, miRNA was extracted from plasma and urine using specimen type-optimized Gu/OcA method with silica-column purification, acid phenol:chloroform with silica columns, and the Exiqon miRCURY biofluids kit. RNA size distribution and integrity were determined using a bioanalyzer. RNA was reverse transcribed and miRNAs were quantified by TaqMan real-time PCR. Proteins in two specimens extracted by Gu/OcA and miRCURY were evaluated by SDS-PAGE.
Summary of Findings:
Optimization of the Gu/OcA protocol to obtain the lowest cycle threshold values for miR-16 and miR-126 identified 0.6M guanidium, 0.5% octanoic acid at pH 4 as the best for plasma and 1.35 M guanidium, 1.5% octanoic acid at pH 4 as the best for urine. Electropherograms showed that the majority of the miRNAs obtained using Gu/OcA were 10-50 nt. The optimized Gu/OcA method was more efficient at extracting miR-16 and miR-126 from plasma than the miRCURY kit (1.65-fold and 2.85-fold, respectively) or acid phenol:chloroform method (7.21 and 2.85, respectively) and from urine than the miRCURY kit (142-fold and not detected, respectively) and acid phenol:chloroform method (2.29-fold and 1.47-fold, respectively). As determined by A260/A280, the miRCURY kit was better at removing proteins from blood than the Gu/OcA method but not as effective at eliminating biopolymers from urine. SDS-PAGE of the supernatant from plasma after treatment with the miRCURY kit showed a 52 kDa protein but there were at least 10 protein bands present after precipitation with GuOcA, indicating that miRCURY kit has a more aggressive protein precipitation. The authors postulate this more aggressive precipitation results in loss of miRNAs through co-precipitation.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform RNA Automated electrophoresis/Bioanalyzer Protein 1D/2D gels Protein Spectrophotometry RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte isolation method Guanidium thiocyanate (0.3, 0.6, 0.9 or 1.35 M)/octanoic acid (0.5%, or 1.5%) pH (4, 5, 6)
miRCURY kit
Acid phenol-chloroform