Circulating miRNA Extraction: A Method to Isolate miRNA from Plasma Samples Via Organic Extraction and Small RNA Enrichment

Abstract

Source: Canale, M. et al., Detection of a Circulating MicroRNA Custom Panel in Patients with Metastatic Colorectal Cancer. J. Vis. Exp. (2019).

This video describes the extraction of circulating microRNA from plasma samples of colorectal cancer patients using organic extraction followed by glass-fiber column enrichment. miRNAs are stable in biological fluids such as serum and plasma, which renders them ideal circulating biomarkers for cancer diagnosis, prognosis, and treatment decision-making and monitoring. This method will help identify new circulating biomarkers capable of predicting clinical outcomes in patients.

Protocol

NOTE: Perform all of the following steps under a sterilized fume hood according to Good Laboratory Practice (GLP).

1. Plasma Collection and Storage

1. Collect 3 mL of peripheral blood sample in a K3E EDTA tube.
2. Centrifuge the tube at room temperature at 1,880 x g for 15 min.
3. Recover the supernatant plasma in aliquots of 450 µL.
4. Store the samples at -80 °C until use.
   NOTE: Process the peripheral blood tube within 2 h of collection. When recovering the plasma from the tube, do not disturb the lymphocyte layer....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Rnase-free Safe-lock 1.5 mL tubes	Eppendorf	0030 123.328
Rnase-free 1000 µL tips	Starlab	S1122-1830
Rnase-free Safe-lock 2 mL tubes	Eppendorf	0030 123.344
Rnase-free 20 µL tips	Starlab	S1123-1810
Rnase-free 200 µL tips	Starlab	S1120-8810
mirVana PARIS RNA and Native Protein Purification Kit	Thermo Fisher	AM1556
100% ethanol anidrous ACS grade	Carlo Erba Reagents	414605
2-mercapto-ethanol	Sigma-Aldrich	M3148
0.2-2, 1-10, 2-20, 20-200, 100-1000 µL laboratory pipettes
Fume hood
Benchtop microcentrifuge
Vortex