JoVE Encyclopedia of Experiments
Cancer Research
0 views • 4:33 min • April 30th, 2023
- 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
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