standard curves sticking together - (Mar/24/2008 )

Hi all,

I am trying to run a real time standard curve from serial dilutions of cDNA using SYBR Green on the Rotor Gene 3000. using actin primers with 2x dilutions are fine and I get a good efficiency and R2. But using my GOI primers the serial dilutions stick together around Ct ~25. My GOI is a low level transcript. is it why the serial dilutions are showing difference in Ct? What's going on?
Thanks for any input

Chris

Hi

Have you checked your melting curves and negative/positive controls? Two options I can think of for your result: a) you're not amplifying your GOI, just primer dimers. You are amplifying contamination (like PCR product).

Hope this helps.

Hi Erica,

I've run the gel for the realtime standard curves, and the band was that of the GOI. So I'm not amplifying primer-dimers. And the NTCs were clean, no contamination. Any suggestions as to what else is at play?

Thanks,
Chris

Theoretically it could mean your PCR components are limiting for the reaction and therefore more concentrated template doesn't produce lower Ct (like it should). You could try to dilute the standards even more (in 10 fold steps) to see if the Cts would start to separate properly.

Hi and thanks all for your ideas.
However, i diluted the standard 5 X and 10X and still the curves stick together. Could it be due to degraded RNA itself or even impure RNA or something?
I checked my RNA by nanodrop and it gave a reading of about 2.2 for absorbance ratio. But only about 20++ ng/ul. But my Cts go up around 20, meaning concentration is not a problem. What is? anyone have this problem before? I even used epicentre's failsafe master mix, but apparently not very failsafe.

Thanks,
Chris

Your RNA concentration is very low, how much of that are you putting in the reaction? With so little RNA, and a low-level expression transcript you might have quite a lot of trouble to amplify it. Any chance you're amplifying gDNA? Have you got -RT in your setup and are they clean?