DNA array - (Oct/17/2001 )
I don't know if it would work for preparing RNA for microarrays, but clontech makes a kit that enriches mRNA's. The whole point of the kit is to amplify these sequences from samples where you need to have more RNA than what can be obtained from the cell/tissue source.
What is the optimum amount of total RNA one can use for the cDNA synthesis for array work?I am working with unstimulated PBMCs and the RNA content is very low as compared to other cells. I get only 25 micrograms from 30 million cells using Qiagen's RNeasy midikit.It is difficult to start with more cells as I have to get more than 30 ml blood from the donors.Thanks in advanceSuparna
in theory a concentration of 1-2 micrograms of total RNA is sufficient. this of course, will also be dependent on the cleanliness of your RNA and how it was generated. it is important to have clean RNA because the presence of contaminants or inhibitors can inhibit the RT (reverse transcriptase). typically a phenol/chloroformstep will be enough to clean it. follow this up with an etoh overnight ppt.
in general, you would want to spot at least 10 ng +/- 10% of DNA for your array.