RNA extraction: Isolation of mRNA

Introduction

This method employs the Dynal® Dynabead mRNA DIRECT kit to extract mRNA from A.fumigatus. The protocol provided by Dynal® was not originally tested in fungi and has been modified for use with A.fumigatus.

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

  • Modified Vogel's minimal medium: Vogel's salts in 1% glucose
  • 0.6 M MgSO4
  • Liquid nitrogen
  • Lysis buffer: provided by Dynal®
  • Washing buffer with LiDS: provided by Dynal®
  • Washing buffer: provided by Dynal®
  • Elution solution: provided by Dynal®

Equipment

All non-sterile containers used for RNA extraction should be baked at 180°C for 8h or soaked for 30 min in 3% H202

  • Buchner funnel and Whatman 54 paper
  • Pestle and mortar
  • 21 gauge needle
  • Dynabeads and magnetic stand provided by Dynal®
  • Glass homogenizer
  • Microcentrifuge and tubes

Procedure

1) Inoculate 50 ml of Vogel's minimal medium to a final concentration of 107 spores per 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 to a 100 mg portion of mycelia and grind in a -20°C cooled mortar to a fine powder - use at least 2 lots of liquid nitrogen. Transfer the powder to a glass homogenizer and add1ml lysis buffer.

4) Homogenize well for 1-2 min and then centrifuge at 5 000 x g for 60 s in a microcentrifuge tube. Remove supernatant to a new tube and press through a 21 gauge needle 3 times. The sample can be stored in liquid nitrogen at this stage.

5) Thoroughly resuspend Dynabeads. Place 0.25 ml Dynabead suspension into a microcentrifuge tube and place on a magnetic stand for 30 s. Remove the supernatant.

6) Add 0.2 ml lysis buffer, resuspend the Dynabeads and place on a magnetic stand for 30 s. Remove the supernatant.

7) Add the A.fumigatus cell extract and resuspend the Dynabeads, incubate for 3-5 min at R.T mixing occasionally. Place on a magnetic stand for 2 minutes and remove the supernatant.

8) Wash the beads twice with 0.5-1 ml washing buffer with LiDS and three times with 0.5 ml of washing buffer without LiDS. Care should be taken to completely remove the supernatant each time.

9) Add 10-20 ml of elution solution and place at 65°C for 2 min. Place the tube on a magnetic stand and transfer the supernatant containing mRNA to a new RNase free tube.

Timetable

Fungal culture Step 1 24 hours
RNA extraction Steps 2 - 9 1 hour

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