A strange phenomenon using differen reagents - (Nov/13/2006 )

Hello everybody,

I have a strange phenomenon with my Real-time results and hope someone can discuss with it.

I use the Promega reverse-transcriptase to get the cDNA from 1μg mRNA. When I do the qPCR using some cheaper master mix such as TOYOBO Sybr Green I master mix, the 200*cDNA works quite well. But if I use some master mix with hot start Taq polymerase such as ABI, Qiagen, the signals were quite weak even with 20* diluted cDNA, it’s detectable very late .

What I tested for is housekeeping gene which should be quite high expressed. I really don’t know the reason.

Best regards

Do you have Rox and is the machine set correctly?

I have a similar problem with my samples too. I use ABI SYBR green and also I use their cDNA archive kit where the RT efficiency is very low too. It doesnt work well for higher conc. of RNA and the RT efficiency for HKG (BAC, B2M) is -3.3 at very low end conc. of RNA (1ng to 1pg), but the RNA conc is not sufficient to amplify Target and hence right now I am doing RT at 60% efficiency.
I will be eager to find out what everyone has to say about this


I have a strange phenomenon with my Real-time results and hope someone can discuss with it.

I use the Promega reverse-transcriptase to get the cDNA from 1μg mRNA. When I do the qPCR using some cheaper master mix such as TOYOBO Sybr Green I master mix, the 200*cDNA works quite well. But if I use some master mix with hot start Taq polymerase such as ABI, Qiagen, the signals were quite weak even with 20* diluted cDNA, it’s detectable very late .

What I tested for is housekeeping gene which should be quite high expressed. I really don’t know the reason.

Best regards[/size]
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