!doctype html> PrimerDigital — Total RNA Isolation Protocol (TRIzol + LiCl)

Total RNA Isolation Protocol

A practical TRIzol-based workflow with LiCl precipitation to enrich for intact total RNA and improve downstream cDNA synthesis. Designed for tissue volumes ~10–200 μl and typical yields ~10–500 μg.

At a glance

  • Room-temperature handling (no ice) for speed
  • Repeatable phase-separation steps for clean aqueous phase
  • LiCl precipitation to remove small RNA & residual DNA
  • Integrity check on non-denaturing agarose gel
Safety note
TRIzol/phenol and chloroform are hazardous. Work in a fume hood, use appropriate PPE, and dispose of waste according to your institution’s chemical safety procedures.
Suitable for
Most animal tissues (including blood) and plant tissues, with rapid mechanical disruption.

Materials

  • TRIzol reagent
  • Chloroform:isoamyl alcohol (24:1)
  • Isopropanol (2-propanol)
  • 80% ethanol
  • 10 M LiCl
  • Fresh Milli-Q water or 1× TE (10 mM Tris-HCl, 0.1 mM EDTA, pH 7.0)
  • 2 ml microcentrifuge tubes
  • Bead/ball for mechanical disruption (optional)

Use fresh solutions and good pipetting practice (aerosol tips, closed tubes). Excessive RNase “treatments” (e.g., poorly prepared DEPC water) can interfere with cDNA synthesis.

Step-by-step procedure

  1. Disrupt tissue. Freeze the sample (e.g., −80°C), then mechanically grind (e.g., bead mill).
  2. Lyse in TRIzol. Add 1 ml TRIzol, vortex thoroughly, incubate 5 min at RT.
  3. Phase separation. Add 0.2 ml chloroform per 1 ml TRIzol; vortex; incubate 3 min at RT.
  4. Spin. Centrifuge at maximum speed for ~5 min at 4°C.
  5. Re-extract aqueous phase. Transfer aqueous phase to a new tube, add equal volume chloroform; vortex; spin 5 min.
  6. Precipitate RNA. Transfer aqueous phase, add equal volume isopropanol; vortex; spin ~10 min at 4°C.
  7. Wash. Wash pellet once with 80% ethanol; spin ~5 min; discard ethanol.
  8. LiCl enrichment. Dissolve pellet (do not overdry) in 1×TE at ~55°C; add equal volume 10 M LiCl; incubate at −20°C (hours/overnight).
  9. Recover total RNA. Spin ~10 min at 4°C; discard supernatant (small RNA & residual DNA). Wash pellet with 80% ethanol; dissolve in fresh water or 1×TE.
Integrity check
Load ~5 μl on a non-denaturing 1.5% agarose gel (with EtBr in-gel) to assess rRNA band sharpness and smearing.

Practical tips

  • Keep tissue volume ≤ 1/5 of extraction buffer volume.
  • Disrupt tissue quickly and completely to minimize degradation.
  • If lysate is viscous (gDNA), extend spin times during extractions.
  • Do not overdry pellets—this slows redissolution and can reduce yield.

Example result

RNA after LiCl precipitation from DNA and small RNA
Figure 1. LiCl plant RNA precipitation. Lanes 1–3: supernatant fraction (DNA + small RNA). Lanes 4–6: RNA pellet after LiCl precipitation.

Related protocols

DNA extraction
Electrophoresis
RNA/DNA preservation
PCR guide & troubleshooting

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