Encyclopedia of Experiments: Cancer Research
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Transcript
- MicroRNAs or miRNAs are small non-coding RNAs that regulate gene expression by degrading mRNA or blocking their translation into functional proteins. Circulating miRNAs are potential cancer biomarkers and are extractable from body fluids like plasma. To begin, mix the plasma with a denaturing solution that simultaneously homogenizes the sample and inhibits RNA-degrading ribonucleases.
Treat with acid phenol-chloroform and centrifuge to separate the sample into two phases, a lower organic phase containing DNA and proteins, and an upper aqueous phase containing RNA. Recover the aqueous phase. Add absolute ethanol to precipitate RNA from the solution.
Bring the sample ethanol concentration to 25% to set an initial condition conducive to large RNA separation. Transfer the mixture to a pre-assembled filter cartridge and centrifuge. Large RNAs selectively immobilize on the filter, while smaller miRNAs collect in the filtrate.
Subsequently, increase the ethanol concentration to 55% to favor miRNA isolation. Transfer the filtrate to a new filter cartridge and centrifuge to trap the miRNAs. Place the cartridge on a collection tube and add a suitable elution buffer. Centrifuge to elute the miRNAs into the tube. In the following protocol, we will show circulating miRNA isolation from the plasma of colorectal cancer patients.
To begin, transfer 400 microliters of the plasma sample into an RNase-free 2 milliliter tube. Then, add 400 microliters of denaturing solution to the tube. Add 4 microliters of 1 nanomolar spike-in.
After mixing the solution, add 800 microliters of acid phenol-chloroform. Vortex the solution for 60 seconds and centrifuge at maximum speed for 15 minutes. Next, transfer for the lysate contained in the upper aqueous phase to a clean tube, taking care not to disturb the interphase.
Measure the volume of the aqueous phase recovered and add 1/3 volume of ethanol to the lysate. Then, place a filter cartridge into a fresh collection tube. Transfer up to 700 microliters of the ethanol lysate mixture to the filter cartridge. Centrifuge the filter cartridge for 30 seconds at 10,000 x g.
Next, measure the total volume of the flow-through. Then, add 2/3 of the volume of ethanol to the filtrate and mix well. Place a second filter cartridge into a fresh collection tube. Then, transfer up to 700 microliters of the sample to the cartridge and centrifuge at 10,000 x g for 30 seconds before discarding the flow-through.
Add 700 microliters of microRNA wash solution to the filter cartridge and centrifuge it with a collection tube at 10,000 x g for 15 seconds. Discard the flow-through and put the filter cartridge back into the same tube.
After this, move the filter cartridge to a new collection tube. Then, add 100 microliters of preheated elution solution to the cartridge. Centrifuge the filter cartridge at 10,000 x g for 30 seconds to recover the microRNAs.
