qPCR Standard Curve - PCR product based

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L.P. Brazil
L.P. Brazil's picture
qPCR Standard Curve - PCR product based

I made all my analyses with a standard curve for beta-actin (control) with purified PCR product amplified from a cell culture gDNA. Afterwards I realized that the cell I used is from mouse and I analyze human samples. I borrowed cells culture from someone. One primers differ 2 bases and other only one base pair. Obviously the amplicons TMs are different too. But the efficiency range from 100 to 96%. So can I use this curve to calculate actin copies in my samples? Or will I have to do it all again?

Ivan Delgado
Ivan Delgado's picture
I would do it again. Just

I would do it again. Just because the efficiency of your mouse curve is good does not mean that it is representative of your human cells. 

On a related note, an efficiency of 96% is not good. Only efficiencies of >99% should be considered good enough. Think of it this way: if every PCR cycle were to only amplify 96 amplicons instead of 100, at the end of 40 PCR cycles you would have a huge difference between a 99% efficiency and a 96% efficiency. I have never used an assay that is less than 99.8% efficient. It is not easy to get an assay to work that well, but at the same time it is not hard. Just design two or three assays and choose the one that works best.

Good luck.

L.P. Brazil
L.P. Brazil's picture
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Hello,
Thanks for your fast answer!!! I had so many problems in this assays. One of them was that storage of Sybr mix. Using the same aliquot of primers and standards for curve and only varying the Sybr stok I realized that 96% efficiencies were archived when I used Sybr stock that were kept on refrigerator for a month. When I keep them in the freezer I got 100%. 
But how about my actin curve construction? 

Ivan Delgado
Ivan Delgado's picture
Hi LP,

Hi LP,

Normally SYBR mixes should be fine in the refrigerator for a month, but you never know. For example refrigerators that are opened often during the day will keep a temperature that is much higher than 4oC in the area closest to the door, so if your SYBR was being stored close to a refrigerator's door, and the refrigerator was being opened often, then your SYBR was not really stored at 4oC for a month but likely experienced as high as >10oC temperatures for at least hours at a time.

If you are getting your assay to work at 100% efficiency using SYBR that was frozen then I would stick to that all the time. 

As far as I can tell your actin standard curve is fine. Standard curves with >99.8% efficiency, and a slope of around -3.32, are good. As I said on my last message though, I would not use a standard curve prepared using mouse DNA to analyze human DNA when the DNA sequence being analyzed in these two organisms is different.

L.P. Brazil
L.P. Brazil's picture
Thank you very much for your

Thank you very much for your help!!!
For sure, I will use this forum any other times.

Best regards, L.P.