Fecal (micro) RNA Isolation

Summary

This protocol isolates high quality total RNA from fecal samples of animal and human subjects. A commercial miRNA isolation kit is used with significant adaption to isolate pure RNA with optimized quantity and quality. The RNA isolates are good for most downstream RNA assays such as sequencing, micro-array, and RT-PCR.

Abstract

It is becoming clear that RNA exists in the gut lumen and feces in animals and humans. The protocol described below isolates total RNA including microRNAs from fecal samples of animal and human subjects. The aim is to isolate total RNA with high purity and quantity for downstream analyses such as RNA sequencing, RT-PCR, and micro-array. The advantages of this optimized protocol in the miRNA isolation are capabilities of isolating highly purified RNA products with additional washing steps described, increased quantity of RNA obtained with an improved method in the resuspension of sample, and important tips of decontamination. One limitation is the inability to process and purify larger sample of more than 200 mg as these sample sizes would cause a difficulty in the clear formation of the interphase. Consequently, the large sample size may contaminate the aqueous phase to be extracted as described in the protocol with organic matters that affect the quality of RNA isolated in the end. However, RNA isolates from a sample of up to 200 mg are sufficient for most of downstream analyses.

Introduction

Extracellular RNA is getting recognized as a significant factor that mediates many biological processes¹. Extracellular RNA in feces was first reported in 2008 as a marker for colon cancer and active ulcerative colitis², and it was recently revealed as a normal component of the gut lumen and feces and mediates host-microbe communications^3,4,5. The purpose of this RNA isolation protocol is to extract high quality extracellular RNA from fecal samples collected from animal and human subjects. The protocol was adapted from a commerc....

Protocol

All methods involving research animals described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Brigham and Women’s Hospital, Harvard Medical School.

All methods involving human research subjects described here are in accordance with the guidelines set by the Partners Human Research Committee.

1. Fecal sample collection

1. Autoclave or prepare a sterile and nuclease-free 2 mL microcentrifuge tube with a screw cap for each animal subject in an experiment.
   1. For human subjects, provide an appropriate, nuclease-free, and sterile stool specimen collectio....

Results

Representative RNAs were isolated from 50 mg mouse fecal samples (2 mouse fecal pellets) and 100 mg human stool specimens respectively and eluted in 50 µL nuclease-free water. Spectrophotometer analysis of the concentration suggests a total amount of 49 µg and 16 µg RNA were isolated respectively (Table 1). The RNA purity was high as indicated by an A260/A280 ratio of ~2.0 and an A260/A230 ratio of ~1.8 (Table 1). As reported³, the majority of RNAs in the feces ar.......

Discussion

It is important to use RNase-free technique to prevent RNase contamination during the isolation⁷. After centrifugation and the formation of a compact interphase, it is key to avoid the interphase, lower phase, and the particle contaminant floating on the top of the aqueous phase when recovering the aqueous phase. Additionally, two washing steps with 500 µL and 250 µL Wash Solution 2/3 are added to eliminate contaminants in the filter membrane for optimized quality. Furthermore, a start s.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acid-Phenol: Chloroform, pH 4.5 (with IAA, 125:24:1)	Thermo Fischer Scientific	AM9720
DPBS, no calcium, no magnesium	Thermo Fischer Scientific	14190-144
Gloves
Microcentrifuge
mirVana miRNA Isolation Kit	Thermo Fischer Scientific	AM1561
Nuclease-Free microcentrifuge tubes (1.5 mL, 2 mL)
Nuclease-Free Water (Not DEPC-treated)	Thermo Fischer Scientific	AM9937
Pipettor and Nuclease-Free Pipette tips (with filter)
PowerLyzer 24 Homogenizer	QIAGEN	13155
RNaseZap RNase Decontamination Solution	Thermo Fischer Scientific	AM9780
Vortex Shaker