Hi,

I doubt that bad RNA quality is the reason. The shifted Cts for GAPDH might be caused by some PCR inhibitor in the samples (Heme, Phenol, ...) or the precense of chelators (-> reduced Mg++ conc).

How do the curves look like? Are they just noisy, do they show normal plateau signal intensities? How do the melting curves look like? How does the gel look like? Do you use internal calibrators (e.g. ROX)? How do the signals in other channels look like (background, ROX, ...)?

Also run control samples where you know that the curves were ok to check if the instrument is still working correctly.

Hi,

what kind of chemistry are you using (probes, SYBR Green, master mix with or without ROX, etc etc) ? Have the reagents been working before properly?

Hi,

It looks like your cDNA is of not good quality based on the GAPDH Ct values which may be due to initial bad RNA quality or presence of inhibitors in this cDNA sample. Which platform and chemistry do you use for your PCR? Have you check the well/ tubes of reactions, was it properly sealed, evaporation.. these may also give such curves.

Are your standards made from the same cells you are investigating?

Sample in excess can also give you this. But have you made a test to see the proper concentration amount of Forward and Reverse primers needed for your reaction? For example, in some of the reactions I do I use 5pMol of Forward primer and 20pMol of Reverse Primer.

Hello All,

I am using Sybr Green Dye chemistry. I ran standards with my primers to check if its the problem with the machine. I got decent curves with my standards , though interestingly I have set up 8 dilutions but I got curves for only three.

Sergio , I used 100nM conc of forward and reverse primers with my standards do I need to change it for the samples ?

I agree with all of you, that cDNA might not be of good quality here, I have re-prepared cDNA and shall run it again today to see if it makes any difference. I am not doubting my RNA samples much because the 260/230 ratio was high enough to have any residual inhibitors.

I was wondering whether it could be that the cDNA is very little in concentration for the machine to detect. I usually dilute the cDNA at a ratio of 1:2 , so I was wondering whether this time when I run should I run undluted ?

Like quitre a few other people I would think there maybe some sort of trace residual that is causing some sort of inhibition. Are you using ROX within your master mix? That may help you get a grips of what is going on. What were the melt curves like at all on the SYBR green traces, may help you see whats going on.

You said you have jagged amplification curves. I thought you meant that you get sigmoid curves, raising at some cycle roughly exponentially and then reaching a plateau, but jaggeld ones, with a large noise component or spikes and scatters. Now I understand that you dont get such amplification curves but just curves that do not show any systematic signal increase over time. Do you get an amplification product? Is the amount of product comparable to the amounts of other products (melting curves, gels)?

Hi Shambhavi, well it's better to perform a test with different concentratiom of primers, it takes some time but with this you can have also a better performance and in some cases spent less primers. By the other way, if your standarts are working well, you can keep the way you are doing.

I think maybe your sample have poor quality but there is the possibility of it being more concentrated than you really need. In some of the experiments I used to do, I used samples previously dosed on NanoDrop, I usually used samples with the concentration of 0,1 to 1,0ng/mL.

I hope this help you.

You can try different conc. of your primers/probe/sample. Also see another discussion that might help http://www.researchgate.net/post/Why_are_my_real_time_PCR_amplification_curves_jagged_regardless_of_using_a_SYBR_green_or_TaqMan_test