If you are looking for a kit RNeasy Plant Mini Kit by Qiagen is the best, though expensive. 

http://www.qiagen.com/products/catalog/sample-technologies/rna-sample-technologies/total-rna/rneasy-plant-mini-kit

No Sir. I am looking for a simple method, I dont want to buy a kit.

The extraction with Trizol (Invitrogen) can be applied to filamentous fungi. The mycelium should be macerated with liquid Nitrogen and then following the steps of extracting Trizol.

Thanks Jose. I will carry out the procedure as you said.

Trizol works of course nicely, but the price is really tremendous for a very very easy recipe. Why not go back to the original article by CHOMCZYNSK and SACCHI in ANALYTICAL BIOCHEMISTRY (1987)? Recently, that technique has been re-discussed in (probably) "Nature Protocols". Buying the chemicals is really much cheaper than ordering the "Trizol" reagent.

Personally, I don't like the phenol treatment too much, and prefer to centrifuge the guanidiniumthiocyanate extract through the traditional CsCl cushion.

Good luck!

Thanks Professor Johannes. But, dont u think the procedure is tedious and let me just ask you, how is the enzyme chitinase used to break down the fungal cell wall as we are finding it difficult to breakdown the same. how can it be used sir?

Tedious is not an easy word. The triazol method takes a few hours (one hour for the invitrogen protocol, four hours in the original publication). Processing the mycelium is always the same, regardless how you extract the RNA afterwards. The CsCl cushion needs approximately 20 hours, but this is done essentially over night. You need, however, an ultracentrifuge and ideally a swinging bucket rotor. If you have this equipment, there is no reason why you should not use it. The machine works for you. If not, the guanidinium/phenol procedure is certainly adequate.

Opening mycelia enzymatically is nearly never a good idea. Guanidinium kations, iisothiocyanate anions plus detergents plus phenol (if you wish) plus 60 degrees temperature are used for immediate inactivation of all enzymatic activities. You should not allow the cells to degrade RNA before you extract it. I recommend to use mechanical opening of cells. Chitinase alone will work only with a few fungi. The amount of chitin can be small, and often it is well hidden beneath protein, glycoprotein, mannan, glucan ...

Good luck - Joh.

Hi Bhargavi

Johannes is right - the CsCl cushion method gives wonderful RNA in large quantites. If you dont have access to an ultracentrifuge with a swing out rotor there are many other methods or kits.

- what do you want the RNA for? How much do you need?

Yes Prof.Johannes and Prof.Paul, we have an access to ultracentrifuge with a swinging rotor. I need the total RNA for c-DNA synthesis, i need it around 5 micrograms. Yes sir, I will go with CsCl cushion method as i want RNA in a quite good quantity and with no DNA contamination for the same. Sir, Though its a basic question, how do you suggest opening of the mycelium. Using a homogenizer or using pestle and mortor, 

... try mortar and pestle under liquid nitrogen. If possible do this under a normal fume hood, in order to keep the vapour with mycelial pieces away from you. This material often has high allergenic potential. Don't let the resulting powder thaw and add it in small portions directly to the lysis mix.

Good luck, jw.

"The following recipe comes from a paper in Fungal Genetics and Biology (42 (2005) 804–812: 4-Dihydromethyltrisporate dehydrogenase, an enzyme of the sex hormone pathway in Mucor mucedo, is constitutively transcribed but its activity is differently regulated in (+) and (-) mating types Christine Schimek, Annett Petzold, Kornelia Schultze, Jana Wetzel, Frank Wolschendorf, Anke Burmester, Johannes Wöstemeyer).

"Sexually stimulated mycelia were harvested into

liquid nitrogen at 0, 20, 40, 60, 80, 100, 120, 150, 180, 210,

and 240min after stimulation and ground to a Wne

powder with mortar and pestle. Control cultures were

sprayed with 20% ethanol (v/v) without trisporoids and

harvested after 240min. For each condition, 10 petri

dishes were used. The ground mycelia were transferred

into 20ml of lysis buVer containing 4M guanidiniumthiocyanate,

2% (w/v) sarcosine, 0.05M

ethylenediaminetetraacetate (EDTA), pH 7.0, and 5% (v/

v) -mercaptoethanol (Geiser et al., 1980) and incubated

at 56 °C and heavy agitation for 20 min. Cell debris was

removed by centrifugation for 20min at 27,000gmax and

4 °C. The supernatant was layered on top of a 1.5ml

cushion of 5.7M cesium chloride in 0.05M EDTA, pH

7.0, in an 11ml tube and centrifuged at 15 °C for 20 h at

153,000gmax. The resulting supernatant was discarded

and the sedimented RNA was resuspended in diethylpyrocarbonate-

treated water. RNA concentration was

determined spectrophotometrically at D260 nm. The

absorbance ratio at 260/ 280 deWning the purity of the

preparations lay between 1.8 and 2.0."

Thanks so much Prof. Johannes for your precious and valuable information. I will get back to you with results i get.