Why does denaturing solution prevent RNA degradation in plasma?

The denaturing solution homogenizes the plasma sample and inhibits ribonucleases, protecting miRNA integrity during extraction. This step is critical for recovering intact circulating biomarkers from blood fluids.

How does acid phenol-chloroform separation isolate RNA from plasma?

Acid phenol-chloroform separates plasma into phases, with RNA partitioning into the upper aqueous phase while DNA and proteins remain in the organic phase. This enables selective recovery of RNA for downstream miRNA enrichment.

What role does 25% ethanol play in the filtration step?

At 25% ethanol, large RNAs bind to the filter cartridge while smaller miRNAs pass into the filtrate, enabling size-based fractionation. This initial condition depletes abundant RNA species to improve miRNA recovery.

Why is ethanol concentration increased to 55% for miRNA capture?

Increasing ethanol to 55% promotes binding of small miRNAs to the filter cartridge, allowing their retention while contaminants flow through. This step enriches the miRNA fraction prior to elution.

How does elution buffer recover miRNAs from the filter?

Adding preheated elution buffer to the cartridge and centrifuging releases bound miRNAs into the collection tube. This step yields purified miRNA ready for quantification or downstream analysis.

Circulating miRNA Extraction: A Method to Isolate miRNA from Plasma Samples Via Organic Extraction and Small RNA Enrichment

JoVE Encyclopedia of Experiments

Cancer Research

0 views • 4:33 min • April 30th, 2023

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- 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.

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Last updated: 4 July 2026

Circulating miRNA Extraction: A Method to Isolate miRNA from Plasma Samples Via Organic Extraction and Small RNA Enrichment

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