qPCR efficiency - how to improve (May/14/2009 )
I have been running qPCR and have efficiencies about 70-80% on the MJ machiine Opticon2. My RNA is fine (via nano-drop ratios). I have only one peak in the Tm. Ct is around 24. What could be the problem with low efficiency besides degraded RNA? how important is the low efficiency. I am fairly new to this technique.
-tlp-
tlp on May 14 2009, 11:43 AM said:
I have been running qPCR and have efficiencies about 70-80% on the MJ machiine Opticon2. My RNA is fine (via nano-drop ratios). I have only one peak in the Tm. Ct is around 24. What could be the problem with low efficiency besides degraded RNA? how important is the low efficiency. I am fairly new to this technique.
How you get your current efficiency (70-80%)?
If you got it from titration curve and convert it using the slope of curve, your PCR reaction is not optimized to its best condition.
From my previous experience, great RNA ration reading does NOT meaning your RNA is free of PCR inhibitor which can significantly alter your PCR efficiency.
It is important you have your target gene and control gene have similar efficiency.
If the efficiency differs between target and control genes, you can get different ΔCT by select different threshold even you set your threshold within linear range.
I think you know it will become a big headache if you have a large ΔCT variation.
Take time make your PCR efficiency as good as it can. It worth your effort when the numerical difference that you get mainly come from biology and/or treatments, not come from variation of assays.
-wuxx0153-
wuxx0153 on May 15 2009, 01:20 PM said:
tlp on May 14 2009, 11:43 AM said:
I have been running qPCR and have efficiencies about 70-80% on the MJ machiine Opticon2. My RNA is fine (via nano-drop ratios). I have only one peak in the Tm. Ct is around 24. What could be the problem with low efficiency besides degraded RNA? how important is the low efficiency. I am fairly new to this technique.
How you get your current efficiency (70-80%)?
If you got it from titration curve and convert it using the slope of curve, your PCR reaction is not optimized to its best condition.
From my previous experience, great RNA ration reading does NOT meaning your RNA is free of PCR inhibitor which can significantly alter your PCR efficiency.
It is important you have your target gene and control gene have similar efficiency.
If the efficiency differs between target and control genes, you can get different ΔCT by select different threshold even you set your threshold within linear range.
I think you know it will become a big headache if you have a large ΔCT variation.
Take time make your PCR efficiency as good as it can. It worth your effort when the numerical difference that you get mainly come from biology and/or treatments, not come from variation of assays.
I am also new to this type of research. How does one determine PCR efficiency? Is this something I can determine prior to using a real time PCR machine (we have an Applied Biosystems 7300)? How similar do the efficiencies need to be? Thanks for any advice!
And to the OP, I have found that I get good ratios on RNA by nanodrop, but sometimes (about 10% of the time) when I subsequently evaluate the RNA on a bioanalyzer, the RNA is of much poorer quality than the nanodrop ratios would have led me to believe.
-Envennom-
Envennom on May 22 2009, 10:13 AM said:
I am also new to this type of research. How does one determine PCR efficiency? Is this something I can determine prior to using a real time PCR machine (we have an Applied Biosystems 7300)? How similar do the efficiencies need to be? Thanks for any advice!
Unfortunately, you have to run PCR to determine the interaction among template, primers, polymerase and other stuffs in that tube (PCR efficiency).
Efficiency is determined by the titration of standard template (in my PCR I also use it as calibrator in every plate)
The links below explain how it is done.
http://www.gene-quantification.de/efficiency01.html#e-curve
http://www.biomedcentral.com/1471-2180/2/17
A part you should notice is some people use 2.0 as perfect efficiency, which means doubling the amplification fragment for each cycle. But some other use 1.0 as perfect efficient, which indicates 100%.
You can also use software LinReg (it should be free), and software can estimate efficiency of each well.
-wuxx0153-
wuxx0153 on May 26 2009, 02:45 PM said:
You can also use software LinReg (it should be free), and software can estimate efficiency of each well.
Sorry my mistake, the name of software should be LinRegPCR.
-wuxx0153-