RNA extraction: miniprep

Introduction 
The Qiagen RNeasy plant extraction kit can be used to quickly extract total RNA from A.fumigatus. The RNeasy technology uses the selective binding properties of a silica-gel based membrane to extract up to 100 mg of RNA species greater than 200 bases in length

A significant quantity of contaminating DNA can occur on extraction of RNA from A.fumigatus using this protocol and treatment with DNase may be necessary.

Material and equipment listed below are those not provided by the manufacturer.

Materials 
All solutions used to extract RNA should be made up in DEPC-treated water and then sterilised. 

  • Modified Vogel's minimal medium: Vogel's salts in 1% glucose
  • 0.6 M MgSO4
  • Liquid nitrogen
  • DEPC - treated water

Equipment 
All non-sterile containers used for RNA extraction should be baked for 8 h at 180°C or soaked in 3% H2O2 for 30 min. 

  • Buchner funnel and Whatman 54 paper
  • Vortex mixer
  • Microcentrifuge and tubes

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 (need ~0.5 g wet weight) 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 microcentrifuge tube. (No more than 0.1g powder is recommended for each miniprep. column)

4) Follow the protocol according to the manufacturers instructions. 

Timetable 

 

Fungal culture Step 1 24 h
RNA extraction Steps 2 - 9 2 h

Tips and general comments 
1) The quality of RNA resulting from this protocol is not of the highest quality and we would not recommend it for library use. The RNA should be suitable for RT-PCR analysis provided all contaminating DNA is removed. We have used RNA extracted in this manner for differential display and Northerns.

2) The kit provides two disruption buffers one containing guanidium isothiocyanate and the other containing guanidium hydrochloride. The manufacturers directions suggest that guanidium isothiocyanate and secondary metabolites produced by filamentous fungi may result in solidification of the sample. However, the buffer containing guanidium isothiocyanate more potently disrupts tissue and is therefore preferable. We have found that the guanidium isothiocyanate buffer in A.fumigatus and A.nidulans did not cause sample solidification and therefore could be used to extract RNA from these fungi.

3) We would recommend caryring out a second elution step (Step 9 in manufacturer's protocol). A significant amount of RNA should be eluted. 

References 
Vogel, H.J. (1956) A convenient growth medium for Neurospora (medium N). Microbiol. Gen. Bull. 13, 42 - 44