High Ct/Cq values in my NO-RT and NTC samples - (Jan/16/2014 )

Hi Bioforum.

I'm currently trying to compare the expression level of two different genes from fermentation samples, however I have had a lot of trouble in getting clean samples for the qPCR.

I use NO-RT and just Supermix, water, primer as controls.

In my most recent try my NO-RT and NTC samples showed Ct values at 37-52 (Im currently running around 60 cycles, not sure if thats to many (additional question?)). Can i neglect these values since they are fairly high? My samples are around 16-21 Ct. Or how would I check if these values have an impact on my samples? Do I need a positive control sinec I just want to measure the ratio between the two genes?

Best regards

Jonas

Most people would consider 40-45 cycles adequate.  If you consider how PCR works to amplify a target, assuming 100% efficiency, every 10 cycles a single copy will be amplified about 1000 times (2ⁿ where n= number of cycles).

Reactions that are appearing over about 35 are probably suspect and could easily be random non-specific amplification.  Certainly anything beyond 40 cycles could be ignored.

Okay, thanks. Lets say all my samples provide a Ct of 35 can you then conclude that you didnt have any success in isolation RNA in the first place or would you have to check with a positive control?

Perhaps - you would need a positive control ( you could do a standard curve too) to test this.  Other problems could be inefficient reactions, inhibitors in the reaction, degraded RNA, reactions not prepared properly...