By Palmer Owyoung
Updated Aug 30, 2022

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The genetic code of a cell resides in its DNA, which remains locked in the nucleus. To study gene expression or to generate complementary DNA (cDNA) libraries, the DNA must first be transcribed into messenger RNA (mRNA). Isolating high‑quality mRNA is essential for detecting rare transcripts, designing microarray probes, and constructing cDNA libraries.

Isolation of mRNA From Total RNA

Step 1 – TRIzol Homogenization

Begin by resuspending pelleted cells in TRIzol Reagent (Life Technologies) or an equivalent phenol‑guanidine solution such as Ambion’s TRI Reagent. This reagent simultaneously lyses cells, denatures proteins, and protects RNA from enzymatic degradation.

Step 2 – Total RNA Isolation

Perform a series of centrifugations to separate cellular components into distinct phases. Discard the yellow, fat‑rich top layer. Collect the red aqueous phase, which contains the complete RNA population (mRNA, tRNA, rRNA, and non‑coding RNAs). Follow the phenol‑chloroform extraction protocol, then wash the RNA pellet with isopropanol and ethanol. Add RNase inhibitors throughout to preserve RNA integrity.

Step 3 – mRNA Extraction

Commercial kits provide the most reproducible mRNA purification. Popular choices include Invitrogen’s FastTrack 2.0 and Ambion’s Poly(A)Pure mRNA Isolation Kit. A typical kit protocol proceeds as follows:

• Combine up to 300 µL of total RNA with the kit’s RNase‑inhibited lysis buffer.
• Heat at 65 °C for 5 minutes, then snap‑cool on ice.
• Add 0.5 M NaCl and dissolve the supplied oligo‑dT (oligodeoxythymidylic acid) into the mixture.
• Centrifuge and recover the supernatant; bind the mRNA to the provided silica matrix.
• Wash repeatedly with low‑salt binding buffer and then with wash buffer.
• Elute the mRNA into the specified volume (typically ~500 µL).
• Precipitate the eluate with sodium acetate and ethanol, then resuspend in 10–20 µL DEPC‑treated water.
• Store at –80 °C and assess concentration and purity using a UV spectrophotometer.

Things Needed

• Laminar flow cabinet
• Personal protective equipment (lab coat, gloves, eye protection)
• Pipettes, tips, biohazard disposal containers
• High‑speed centrifuge, heat block
• RNA‑free bench surfaces
• RNAzap or RNAoff reagents
• Commercial mRNA extraction kits (Invitrogen, Ambion, etc.)
• Sodium acetate, ethanol, isopropanol
• DEPC‑treated water
• UV spectrophotometer and consumables
• Cells suitable for RNA extraction
• TRIzol or TRI Reagent
• RNase inhibitors

TL;DR

Keep all reagents, cells, and extracted RNA cold by placing them on ice. Cold conditions inhibit RNase activity released during homogenization.

Warning

TRIzol and similar phenol‑guanidine reagents are toxic. Avoid skin and mucous membrane contact and adhere strictly to institutional safety protocols.

References

• “cDNA Library Protocols”; Ian G. Cowell and Caroline A. Austin, 1997
• “In Vitro Transcription and Translation Protocols”; Martin J. Tymms, 1995