Trouble with standard curves for real-time PCR - (Oct/14/2005 )
I have a weird problem recently.
I used to work with RT-PCR using Sybr Green Dye. For each new primers, a serial dilution of 100, 10, 1, 0.1, 0.01 ng of cDNA template was used to generate a stardard curve. I always got a perfect stardard curves until recently. The Ct differences between the 10 and 1 ng templates are always around 6 instead of 3.3 and Cts from subsequent concentrations are also disordered. But the differences between 100 and 10ng are consistant, around 3.3.
I have tried different kits from different vendors, changed all other reagents like water and all tubes and tips, but this problem still exists regardless what templates and primers I use. I have tested samples from human, mouse and rats with more than 10 different primers. Also did machine (ABI 7700) background and dye calibration. But nothing helps.
Does anyone have similar experience? Any suggestion is appreciated.
I used to work with RT-PCR using Sybr Green Dye. For each new primers, a serial dilution of 100, 10, 1, 0.1, 0.01 ng of cDNA template was used to generate a stardard curve. I always got a perfect stardard curves until recently. The Ct differences between the 10 and 1 ng templates are always around 6 instead of 3.3 and Cts from subsequent concentrations are also disordered. But the differences between 100 and 10ng are consistant, around 3.3.
I have tried different kits from different vendors, changed all other reagents like water and all tubes and tips, but this problem still exists regardless what templates and primers I use. I have tested samples from human, mouse and rats with more than 10 different primers. Also did machine (ABI 7700) background and dye calibration. But nothing helps.
Does anyone have similar experience? Any suggestion is appreciated.
Dear junjunxu,
From my experience, I think this is a typical degradation problem. Either yous standard cDNA or your primer is degraded.
1. Could you please tell me how you prepare your cDNA standard?
2. Are you prepare a cDNA stock (know amount) and you just serial dilute it prio PCR or you serial diluted them first and store at -20oC?
Best regards
I have a weird problem recently.
I used to work with RT-PCR using Sybr Green Dye. For each new primers, a serial dilution of 100, 10, 1, 0.1, 0.01 ng of cDNA template was used to generate a stardard curve. I always got a perfect stardard curves until recently. The Ct differences between the 10 and 1 ng templates are always around 6 instead of 3.3 and Cts from subsequent concentrations are also disordered. But the differences between 100 and 10ng are consistant, around 3.3.
I have tried different kits from different vendors, changed all other reagents like water and all tubes and tips, but this problem still exists regardless what templates and primers I use. I have tested samples from human, mouse and rats with more than 10 different primers. Also did machine (ABI 7700) background and dye calibration. But nothing helps.
Does anyone have similar experience? Any suggestion is appreciated.
Dear junjunxu,
From my experience, I think this is a typical degradation problem. Either yous standard cDNA or your primer is degraded.
1. Could you please tell me how you prepare your cDNA standard?
2. Are you prepare a cDNA stock (know amount) and you just serial dilute it prio PCR or you serial diluted them first and store at -20oC?
Best regards
Thanks for your reply.
For cDNA standard, I did serial 1:10 dilution of cDNA template just prior to PCR in DNAse and RNAse free water (from invitrogen). Primers were pre-dissolved in DEPC water (from Ambion) to make 100uM stock and 10uM working solutions and kept in -20c.
Since I have tried several cDNA freshly synthesized from differenc sources of total RNA and PCRs were peformed within 1-2 days, the chances of cDNA degradation are pretty low.
Do you think cDNA could be degradated during PCR preparation if the water contains DNAse?
how long ago did you aliquot your primers?
I bet they are degrading there in the ol' -20
Dear junjunxu,
How did you qauntify you cDNA?
Did you carry out RT step-->purify-->qauntification? Or just quantify right after RT step?
Did you quantify your cDNA every time before serail dilute them?
Two possibilities:
1. If you quantify your cDNA with out purification, or you quantify your cDNA every time before, you might end up with over estimate your initial amount of cDNA, therefor you standard curve is disordered.
2. aimikins is right. Your primer is degraded in -20oC. If that is the case add more primer or resynthesized your primer.
By the way, what is the primer concentration that you use in your assay?
Best regards
Hi! I have the same problem. I do my real time standar curve for the first time, and it work fine, but after a few frozen and defrost it doesn't work anymore. I have my primers at a concentration of 20 micromolar.
Usually I do the RT in a 96 well plate, then I dilute the samples 1/10, and use 2-5 microliters in the real time. I would try to do a new standar curve using the same primers dilution and a new one.
Another Idea? and what can I do if my cDNA samples are being degraded?
Thanks!
Keep stocks of primers probes at 100uM and aliquot.