taqman high CT's - (Aug/06/2004 )
The taqman I ran has very high CT's. Even RPL19 is very high. I checked the RNA concs again and everything seems fine. What else could be causing these high CT's? I am clueless. Thank you for any help
How many different primer sets have you used? Maybe you're reading on the wrong wavelength. We were having the same problem, granted it was DNA, and found out the fluorescence was off. Hope that helps.
when our RT lost it's activity, i got high CTs, too. i found out that they correllated to not DNaseI digested DNA copys in the RNA prep. There always were 40-100 copys of DNA left in the sample, ever after digestion. They gave me positive signals in taqman assays at >38 cycles...
so maybe your sample doesn't hold rna or the RT reaction won't work, for whatever reasons...
I had the same problem. In my case, I change the Retrotranscription kit and now it is alright. Sometimes the kit to retrotranscribe RNA to cDNA is not efficient enough. You can try another kit, you can ask me if you want, for the one I am using. I don't want to do free publicity!
what do you mean by 'very high'?
one of my genes usually runs about 30-32
the other 3 run about 18-22 depending on conditions
it's all relative
Please check these few things:
1) run your PCR product on gel to confirm.
--> if you see a nice band but your CT value is high, something wrong with your probe.
--> if you dont see a nice band, you PCR is problematic, optimised your PCR.
2) what reporter and quencher are you using?
--> make sure that your reporter and quencher are in the cirrect match and read by the correct
3) RT loss activity could be one reason.
--> Is your kit provide any control?