Microarray for banana - (Jan/11/2008 )
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. Has anybody done a microarray expt with babana RNA? Can somebody suggest something to extract RNA from banana leaves and get rid of polyphenolics effectively....thanks in advance
Unfortunately, I've never tried the banana leaves; however, I also had problems with labeling. There are few standard approaches that will let you improve it.
#1 Take less RNA for RT. It will reduce concentration of the inhibitors if they are present.
#2 Take more "nucleotides". Velocity of a chemical reaction often grows with the concentration of the substrates.
#3 Take more labeled substrate. It will increase probability of the incorporation.
#4 Take more enzyme. Velocity of the enzyme reaction is proportional to the concentration of the enzyme.
#5 Change the Kit. Ambion (Applied Biosystems) is not the only company on the market. In my knowledge, Qiagen also has an RNA kit for plants.
#6 Keep right proportion of TRIZOL and the tissue. Take less tissue if you feel that TRIZOL is overloaded and your samples become contaminated.
#7 Run three parallel reactions instead of one. Few extra tubes will increase your chances to get the right numbers.
#8 Concentrate your samples before you proceed to the quantification analysis. Higher concentration of the fluorophore will decrease noise and minimize the error.