I have extracted using Trizol reagent and RNAeasy Kit of Quiagen, but until now I couldn't get an RNA clear and in great quantity.
For giving hints its helpful to know your experimental procedure. At the moment it can be anything from not enough fungi over bad extraction over to bad reagents.
It would be helpful if we know a bit more about the state of the fungi when your extracting. I try to grow them so that they make less polysaccharides etc. If you have a good growing saprophyte you can try to grow them on agar with cellophane between the medium and inoculum to ahve an easy peel layer of mycelium. If you have to harvest from liquid culture, filter and squeeze out as much as possible, eg through miracloth.What I found to be crucial is good grinding of the material, extremely good pulverisation helps the extraction.Use a cold beat beater or ball grinder (dry ice) or mortar and pestle with a bit of super clean sand and liquid nitrogen. What do you need it for? Norhterns need a lot but not super clean and qPCR needs superclean but not that much.
how do you check your RNA purity? nanodrop spectrum?
if its not clean enough, just percipitate your rna with isopropanol and wash the pellet - repeat percipitation until you make it pure enough.
I've tried grow them with cellophane, however this didn't work. I use carrot-agar solid medium and mycelium of five days grow.The mycelim was lyophilized with liquid nitrogen, and I use 100 mg of this powder for extraction.
For check the purity of RNA I use nanodrop spectrum, agarose gel 1% and Bioanalyzer and all showed amounts carbohydrate and protein.
For precipitation i've tried isopropanol, absolute alcohol and lithium chloride and I already tried precipitate, than pruficication with chloroform and precipitate again. The Proteine and carbohydrate are gone, as well, as RNA.
you can try the hot phenol-LiCl method by pathak and lochab. u can search for the detailed protocol in canadian journal of microbiology
I used the RNeasy from Qiagen mentioned by you for RNA isolation from Penicillium chrysogenum during my PHD. I found it very helpful for isolation which I later used for mRNA quantification using Real time PCR, I have published a paper based on this work which you can find in my profile. Especially in case of fungal culture the most difficult step is the breaking of the cell wall. During my research the initial cell breaker column from Qiagen kit was sufficient for the lysis of the cell. But later a colleague of mine bought a FastPrep®-24 Instrument for efficient cell lysis of her fungal culture, for tough to break cell wall. I think that the limiting step in RNA isolation is breaking the cell. And if you want to do post-processing and quantification of your RNA I would strictly suggest kits to make your life easy.
All the best !!!
RNeasy kit from Qiagen will give high pure RNA for further experiments (e.g PCR)
Pity the cellophane didn't work, it has done wonders for me. Make sure it is proper cellophane (transparent cellulose) and not PE or other plastics that in some languages is colloquially is called like that. Cut it in pieces that fit in your plates and autoclave between any filter paper (wet is OK).
What do you mean with "The mycelim was lyophilized with liquid nitrogen, and I use 100 mg of this powder for extraction."?Freeze dried or flash frozen? try to get rid of all the agar and liquid, the ice will make it harder to break the cells open in some systems. Never let it thaw after cell-rupture without the extraction buffers that have Phenol / SDS and/or guanidinium isothiocyanate .
I would go for trizol first, incubate the mycelium powder at elevated temperature and give it an extra spin before mixing with Chloroform (or BCP). ncubate 1 hr at 30°C in shaker, Centrifuge at max 12000 x g for 10 minutes at 4°C to remove extracellular material and polysaccharides, transfer supernatant to fresh eppie.
You can even put it on an RNAeasy column after the whole isolation to get ridof some more contaminants.......
Here is the paper. This method has been tested in yeast, plants and blue-green algae till now.
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