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.
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.
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 communications3,4,5. 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 commercial miRNA Isolation Kit. The RNA acquired is utilized for downstream analyses such as RNA sequencing, RT-PCR, and micro-array. The protocol includes several important and useful tips to maximize the quantity and quality of RNA found in the feces of animals and humans. The reason to develop and optimize this method of RNA (including microRNA) isolation is to decrease microbial RNA in the feces, limit the variables in the research studies and analyze the RNA composition in the gut without accounting various confounding factors and sources of contamination. Of note, this RNA isolation minimizes the release of RNA from living cell and living microbes (cellular RNA). It focuses on extracellular RNAs that have been released by gut cells or been acquired via food intake. Principally, this method is not suitable for studies where the microbial transcriptome is investigated.
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
2. Preparations of wash solutions
3. Preparations of equipment and materials
4. Feces resuspension
5. Organic extraction
CAUTION: Use the hazardous chemical fume hood for the following steps until Step 6 with the use of acid-phenol: chloroform and ACS grade 100% ethanol due to their toxicity and inflammability. Change PPE as needed and follow proper standard precautions when dealing with hazardous material.
6. Final RNA isolation
7. Elute RNA with 50 µL nuclease-free water
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 are microRNA and those microRNAs can exist in the exosome. Consistent with this, a chip-based electrophoresis assay of RNA suggests that representative RNA isolates from mouse and human feces are low in or lack of 18S and 28S rRNA compositions, and the size of RNA isolates falls in the small RNA region (Figure 1A). A further small RNA electrophoresis with the chip-based electrophoresis reveals that a large portion of the RNAs are of microRNA size (Figure 1B). This is consistent with the observation that the quantification completed by using a small RNA bioanalyzer is comparable to that obtained with previous assays6.
Sample ID | Elution volume (µL) | RNA concentration (ng/µL) | A260/A280 | A260/A230 | Yield (ng) |
Mouse | 50 | 978.333 | 2.036 | 1.897 | 48916.65 |
Human | 50 | 330.759 | 1.981 | 1.849 | 16537.95 |
Table 1: Representative nanodrop analysis of RNA isolated with this protocol. Representative RNAs were isolated from 2 mouse fecal pellets or 100 mg human stool specimens, eluted in 50 µL nuclease-free water. RNA concentration, ratio of A260/A280, and ratio of A260/A230 were measured with nanodrop.
Figure 1: Representative chip-based electrophoresis analyses of size distribution of fecal RNA isolates. (A) Representative RNAs isolated from 2 mouse fecal pellets (left panel) and 100 mg human stool specimens (right panel) using the protocol described here were characterized using the chip-based electrophoresis assay. This assay suggests that the majority of RNA isolates were small RNA. (B) The isolates were then subjected for the small RNA electrophoresis with the chip-based electrophoresis system to analyze the size distribution of the isolates. Please click here to view a larger version of this figure.
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 sample material of more than 200 mg is not recommended as it may create difficulty in the clear formation of an interphase. Similarly, a sample material of less than 25 mg is not recommended as it may not be sufficient to extract enough RNA samples for downstream analysis.
The incredible growth in microbiome study has driven the measurements of microbial species, genes to metatranscriptional studies of the microbial profile8. MicroRNAs in the stool have been studied as markers for diseases9,10,11. Since the first report of fecal microRNA mediating host-microbe interactions3, increasing studies start to investigate the contributions of host and diet in the gut ecosystem12,13,14. Noteworthily, due to the richness of microbes in the gut lumen and feces, studies focusing on host arm of the host-microbe interaction demands minimal RNA contamination from microbes. Thus, an RNA extraction protocol that includes steps of cell lysing15 is not ideal for the study of RNAs released from host and diet. As such, we adapted this protocol to eliminate lysis steps for minimizing contaminations of RNA from living bacteria and living host cells in feces.
This protocol works for studies where extracellular RNA in the fecal or gut lumen content is an aim of interest. RNA isolated using this protocol is total RNA, including microRNA as major component. This protocol does not distinguish whether the RNA is in exosome, microvesicles, or in a vesicle-free form.
The authors have nothing to disclose.
We received technical assistance from the Biopolymers Facility at Harvard Medical School for bioanalyzer. This work was supported by National Multiple Sclerosis Society research grant RG-1707-28516 (H.L.W. and S.L.).
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 |