generating cDNA from human brain RNA - (Jan/05/2011 )
I have extracted some RNA from human brain samples and want to generate cDNA from this. I prepared cDNA using random hexamers, oligo(dT) and gene specific primers. Next I try to amplify the genes of my interest, but do not get any product after PCR. All i see is the low molecualr weight primers or primer dimers.
For gene specific primers I used 2 ul of 1uM antisence primer in a 20 ul reaction volume and i do RT for 50 min at 55 C.
The genes of my interest range in size from about 1Kb to about 6 Kb.
Any suggestions as to what I am doing wrong or what else I can try.
Did you check the quality of your cDNA by amplifying a housekeeping gene or any other gene already established in your lab?