Help - RNA extraction from banana leaves - (Jan/08/2008 )
I am trying to isolate RNA from banana leaves for a microarray experiment. I tried extracting with trizol which gave me high protein and salt contents (my 260/180 and 260/230 was quite poor). I did a bioanalyzer profiling of the samples and could see that lot of DNA was also carried over...I also tried extraction using MirVana kit (from Ambion) and got a good profile RNA but when i proceeded to the microarray labeling step, it miserably failed. The dye incorporation was almost zero. Can somebody suggest something to extract RNA from banana leaves and get rid of polyphenolics effectively....thanks in advance
Some time ago, I had seen RNA prep protocols specific for banana. There is more than one in journals like plant molecular biology reporter, biotechniques, analytical biochemistry. You can easily find them searching on the journals, or even with google. I suggest you to try at least one of them. If the protocol is for fruits and you use it for leaves it's probably better to reduce the amount sample, lowering the sample/buffer ratio.
You can also try the CTAB method for which you can find reference on the topic 'rna extraction from flowers' on the botany forum.
Trizol sounds like the worse option to me, but if you had high yield you can try to clean up that rna by repeated LiCl precipitation, and perhaps a final passage on some column (like quiagen).
Thank you Andrea, I had been looking through many of the protocols. I found a protocol for RNA extraction from cotton tissue at http://www.eeob.iastate.edu/faculty/Wendel...aextraction.htm ....I'm going to give it a shot...but it seems quite laborious compared to standard methods of extraction.
But I'm wondering why MirVana kit had given me a great profile in a bioanalyzer run but no dye incorporation after labeling!!