RNA extraction: large scale prep

Introduction

This method can be used to extract total RNA of high purity from A. fumigatus. The RNA should be of suitable quality for all purposes including primer extension and library construction after further purification of mRNA as well more standard procedures such as Northerns and RT-PCR. There should be little contaminating DNA and the prep. volumes can be varied according to the amount of starting material.

Materials

All solutions used to extract RNA should be made up in DEPC-treated water using dedicated chemicals and sterilised by filtration or autoclaving.

  • Vogel's minimal medium (Vogel, 1956)
  • 0.6 M MgSO4
  • Liquid nitrogen
  • Extraction buffer:
    • 50 mM Tris - Cl pH 8.0
    • 4 % Aminosalicylic acid
  • Acid equilibrated phenol : chloroform (5:1) pH 4.7
  • Chloroform : isoamyl alcohol (24:1)
  • 8M LiCl
  • 0.5 M EDTA
  • Resuspension buffer:
    • 40 mM Tris - Cl pH 7.5
    • 20 mM sodium acetate
    • 5 mM EDTA
    • 1 % SDS
  • 3M sodium acetate
  • 100 % ethanol
  • 70 % ethanol
  • DEPC - treated water

Equipment

All non-sterile containers used for RNA extraction should be baked at 180°C or soaked for 10 min in 3% H2O2.

  • Buchner funnel and Whatman 54 paper
  • 15 ml polypopylene tubes
  • Vortex mixer
  • Bench top centrifuge and microcentrifuge

Procedure

1) Inoculate 50 ml of Vogel's minimal medium to a final concentration of 107 spores / ml and incubate with shaking at 200 rpm until late exponential phase (18-24 h) at 37° C.

2) Dry down the mycelium onto Whatmann 54 paper using a Buckner funnel and a side-arm flask attached to a vacuum pump and wash with 0.6 M MgSO4.

3) Add liquid nitrogen and grind in a -20° C cooled mortar to a fine powder - use at least 2 lots of liquid nitrogen. Add the powder to a chilled 15 ml polypropylene tube.

4) Add 1 ml of ice cold extraction buffer for every 1 ml volume of powder and two volumes of acid-equilibrated phenol : chloroform. Vortex mix for 1 minute and centrifuge at 3000 x g for 10 min at 4° C.

5) Remove the upper phase to a fresh tube and repeat the phenol : chloroform extraction.

6) Remove the upper phase to a fresh tube and add two volumes of chloroform : isoamyl alcohol. Mix and centrifuge at 3000 x g for 5 minutes, remove the upper phase to microcentrifuge tubes and add LiCl and EDTA to final concentrations of 2 M and 1 mM respectively.

7) Leave O/N at 4° C to precipitate RNA. Centrifuge at 15000 x g at 4° C for 30 min, remove the supernatant and resuspend the pellet in 300 μl of resuspension buffer. Incubate at 37°C for 5 minutes.

8) Centrifuge for 5 min at 15000 x g to pellet undissolved debris and transfer the supernatant to a fresh tube.

9) Add 0.1 x volume of sodium acetate and 2.5 x volume of 100 % ethanol, incubate at 4° C for 30 min and centrifuge at 15000 x g for 30 min at 4°C.

10) Remove the supernatant and wash the pellets in 70 % ethanol. Air dry the pellets and resuspend in DEPC-treated water. Store RNA samples at - 80°C

Timetable

 

Fungal culture Step 1 24 hours
RNA extraction Steps 2 - 10 5h with one overnight step

Tips and general comments

1) It is very important to keep the samples as cold as possible to ensure that the RNA remains intact. This is especially true during the grinding of the mycelium. All utensils coming in contact with the mycelium are pre-chilled using liquid nitrogen

2) The growth conditions given here are generalised to ensure a reasonable yield. It is probably advisable to avoid cultures that have entered stationary phase where the presence of nucleases might lead to problems with degradation.

References

Vogel, H.J. (1956) A convenient growth medium for Neurospora (medium N). Microbiol. Gen. Bull13: 42-44