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Back to Eccles Lab > DGG Protocols

Contents

Two-step RNA extraction by using Tri Reagent and columns

Introduction

This is a tried and true protocol that is pretty much bullet-proof if performed as specified. The combination of RNA extraction from Tri Reagent (MRC) or Trizol (Invitrogen) homogenates combined with a column-based clean-up gives good quality RNA that is relatively free of inhibitors and works well for downstream application such as reverse transcription and RNA amplification for array work. We certainly have evidence that RNA isolated by this method works more reliably in downstream applications than Tri* alone.

General Guidelines

For this protocol we follow the first two steps of the Tri* protocol - Homogenisation and Phase seperation - and then feed the resulting aqueous phase into a column-based clean-up.

Materials Needed

Procedure

Step 1 - Tri Reagent

Homogenisation

Phase seperation

  1. Store the homogenate for 5 minutes at room temperature to permit the complete dissociation of nucleoprotein complexes.
  2. Supplement the homogenate with 0.1 ml BCP or 0.2 ml chloroform per 1 ml of TRI Reagent, cover the samples tightly and shake vigorously for 15 seconds.
  3. Store the resulting mixture at room temperature for 2-15 minutes and centrifuge at 12,000 g for 15 minutes at 4 dC.
  4. Following centrifugation, the mixture separates into a lower red phenol-chloroform phase, interphase, and the colourless upper aqueous phase. RNA remains exclusively in the aqueous phase whereas DNA and proteins are in the interphase and organic phase. The volume of the aqueous phase is about 60% of the volume of TRI Reagent used for homogenisation.
  5. Transfer the aqueous phase to a fresh 1.5 mL tube and save the interphase and organic phase at 4 dC (-80 dC long-term) for subsequent isolation of DNA and proteins.

Step 2 - Column clean-up

  1. Add an equal volume of 70% ethanol to the Trizol aqueous phase.
  2. Vortex for 10 seconds.
  3. Transfer to column.
  4. Follow column manufacturer's instructions.