No RT control showed strange results - (Jun/29/2006 )
I transfected a gene into T84 cells. GFP control showed transfection effeciency is good. So I then tested gene expression using real-time PCR.
My no RT control was done by following normal cDNA synthesis protocol, but bo RT was added.
I would expect nothing come up in no RT control. But the fact is: In my no RT control, my reference gene (CyPA) didn't come up while my transfected gene came up at exactly the same cycle as the RT synthesized cDNA sample.
It is plasmid DNA contamination?
I used trizol protocol to extract RNA. Is trizol protocol can separate RNA and plasmid? which step is doing that separation?
Any other suggestions?
Did you DNase treat your RNA before cDNA synthesis?
Thanks for the reply. I then did the DNAase treatment. But I suffered a great RNA loss. My RNA was 5 times less than before and become not enough for cDNA synthesis. So I use glycogen to concentrated it and resuspended in 18ul water, then use 9ul for cDNA synthesis and 9ul for no RT control. My cDNA synthesis might worked because the PCR still give signal. But my Ct value is a lot later. Before my gene come up at cycle 15. Now it come up at cycle 20, and no RT control come up at cycle 29.
Is cycle 29 late enough? What cycle was your no RT control come up?
i tend not to trust any negatives if the Ct is less than 30, hell some of my genes of interest come in around there!