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Total RNA extraction based on acid guanidinium thiocyanate-phenol-chloroform method.md

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Total RNA extraction from mononuclear cells based on acid guanidinium thiocyanate-phenol-chloroform method

Table of contents

Equipment

  • 1.5 ml tubes;
  • set of automatic pipettes ranging from 1 µl and 1 ml;
  • centrifuge.

Reagents

  • 80% EtOH;
  • Trizol (Invitrogen) or QIAzol (QIAGEN), or other analogous products;
  • steril RNAse free water;
  • chloroform;
  • isopropanol;
  • Pellet Paint® co-precipitant (optional).

Procedure

1| Add 1 ml of Trizol to your suspension of mononuclear cells and pipette extensively for the liquid to become transparent.

CRITICAL STEP: Improper ratio Trizol reagent: sample may lead to insufficient cell lysis and affect the RNA yield. Use correct amount of lysis reagent. For example, use 1 ml of Trizol per up to 10^7 cells.

2| Add 200 µl of chloroform. Shake well.

3| Centrifuge at max speed for 20 minutes at +4.

4| Transfer the tubes on ice. While keeping the tube cool, transfer the upper phase into a new tube.

5| Add 1 µl of co-precipitant (optional) and 500 µl isopropanol.

6| Centrifuge at max speed for 20 minutes at +4.

7| Discard the supernatant.

8| Add 200 µl 80% EtOH.

9| Centrifuge at max speed for 2 minutes at +4.

10| Repeat steps 7-9 (optional).

11| Discard the supernatant.

12| Place the tube opened at 50 degrees. As soon as EtOH evaporates take the tube off the thermostat.

CRITICAL STEP: Do not overdry RNA - it leads to a decrease in recovery.

13| Dilute RNA in steril RNAse free water.

14| Check the amount and quality of RNA using gel-electrophoresis.