We present a protocol for isolating RNA from fecal samples to study microRNAs. This protocol can be used to isolate RNA from feces with high quality and quantity. It minimizes RNAs from living microbes.
It is important to emphasize that and the binding buffer is not used in this protocol. This protocol is simple and straightforward. Begin by resuspending 25 to 100 milligrams of fecal samples in 600 microliters of sterile 1X DPBS in a two-milliliter microcentrifuge tube.
Incubate the tube containing the sample at room temperature for 30 minutes. Then mash the mixture with a one milliliter pipette tip and vortex well. Resuspend the mixture in a homogenizer with a setting for one cycle at 4, 000 rotations per minute for 45 seconds to optimize and increase the quantity and quality of RNA.
Add 600 microliters of acid phenol chloroform solution to the sample and vortex the mixture for 60 seconds. Alternatively, to optimize an increased quantity of RNA in the yield, mix it with a homogenizer. Centrifuge the sample for 15 minutes at 10, 000 x g at room temperature to separate the aqueous and organic phase.
Repeat the centrifugation until the interface is compact. Transfer the upper aqueous phase carefully without disturbing the lower phase to a new two milliliter microcentrifuge tube with a hinge cap. Add ACS grade 100%ethanol at 1.25 x the volume of the acquired aqueous phase and vortex for three seconds.
Place the filter cartridge into the collection tubes. Load 600 microliters of each of the samples in the filter cartridge provided in the microRNA isolation kit. Centrifuge at 10, 000 x g for 90 seconds to filter the mixture through the cartridge.
Then discard the filtrate. Repeat this until the entire volume of the mixture is filtered through the same filter membrane in successive applications. After all the sample is filtered.
add 700 microliters of microRNA wash solution 1 into the same filter cartridge. Centrifuge for 60 seconds to filter the wash solution through the filter cartridge. Discard the filtrate and place the filter cartridge on the same collection tube.
Wash the filter cartridge with 700 microliters of wash solution 2/3 prepared in ACS grade 100%ethanol and centrifuge at 10, 000 x g for a minute. Discard the obtained filtrate and place the filter cartridge into the same tube. Wash the filter cartridge with 500 microliters of wash solution 2/3 and centrifuge at 10, 000 x g for a minute.
Discard the obtained filtrate and place the filter cartridge into the same collection tube. Wash the filter cartridge with 250 microliters of wash solution 2/3 prepared in ACS grade 100%ethanol and centrifuge at 10, 000 x g for a minute. Discard the obtained filtrate and place the filter cartridge into the same collection tube.
Finally, transfer the filter cartridge into a new collection tube and spin the assembly for five minutes to remove the residual fluid. Transfer the filter cartridge into a new collection tube then add 50 microliters of nuclease-free water to the center of the filter and cap the tube. Incubate the tube at room temperature for 10 minutes.
Then spin the tube for five minutes at 8, 000 x g to recover the RNA into a new collection tube. 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. A further small RNA electrophoresis with the chip-based electrophoresis revealed that a large portion of the RNAs are of microRNA size.
The purity of the extracted RNA was high as indicated by the absorbance ratio of 2.0 for 260 and 280-nanometer wavelengths and 1.8 ratio value for absorbance at 260 and 230 nanometers. To ensure success of the organic extraction, make sure that the faces are obvious and only transfer the upper aqueous phase without disturbing the lower phase, even at the cost of a reduced volume of our aqueous phase.
