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real time qRT-PCR and low expression gene - (Sep/12/2011 )


Your advice would be much appreciated. One of the genes I am working with (imprinted gene) is expressed at extremely low levels, so reat time qRT-PCR fails to detect any transcripts. Can anyone suggest any other ways to detect few transcripts (other than performing pre-amplifaction)?? Is there any other PCR method (nested, conventional etc) that is more reliable than real time qRT-PCR?

Thank you very much


at what cycle number do you see the expression (start) of this gene ? and upto what amt of starting cDNA (in ng/reaction) have you tried to conclude that it fails to express in standard condition?


thanx for your reply, i see expression after clycle 36.All my samples have standard number of cells (always less than 1800 cells). When I use around 1800-2000 cells I have no expression. If I do the same and use RNA that was purified from >6000-8000 cells I get some amplification but the expression is very very low. But the point is to do the analysis on small amounts of cells