Standard Curve Issues with ABI qPCR - Problems getting good R^2 values and efficiency (May/03/2006 )

Hi, everyone, im new to this forum.

Im a grad student at Rutgers, working on the qPCR. Recently, Im exploring the expression of a receptor in the pig uterus. I'm having some problems with my qPCR.

I've isolated the RNA and it looks good (18 28S bands good, and OD 260/280 = 2) I obtained cDNA using the invitrogen kit, and am now in the process of setting up the qPCR using the ABI Syber Green system. Currently, my problem involves primer validation.

I am running a standard curve at .001X, .01X, 1X and 10X dilutions of cDNA. My R^2 values are very poor (around .6 to .7). Also, I do not have efficiency based on my delta Ct plots(using cyclophilin as my housekeeping gene). The amplification curve looks good, and there appears to be no dimers in my cDNA based on the melting curve. My Ct values are consistent within duplicates.

So my problems seems to reside in generating the standard curve. Does anyone have any ideas as to why the points are not fitting correctly? Are these concentrations reasonable ones to choose for generating standard curves? The problem points are 1X and .01X, at least they seem furthest from the line.

Any help would be greatly appreciated. Thanks!

tjkungfu

We use b-actin or 18S as house keeping gene. I think the reason could be low expression of gene or the standards have not been properly made.

Try again, ask somebody else to make dilutions for you and use other house keeping gene.

Rohit

Hi,

Another possibility might be to use PCR product to make your standard curve. I have been doing this in the last few weeks, mainly because my target gene isn't very highly expressed and my cDNA quantity and quality are both limited.

The advantage is that you can do a much larger dilution range - I have done a 6 or 7 fold dilution series successfully - then you get a more accurate result. I have done 1x, 1/10, 1/100, 1/1000, 1/10000, 1/100000 and it worked really well with PCR product.

good luck