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PCR efficiency important in real time absolute quantification?


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4 replies to this topic

#1 appleyun

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Posted 17 June 2009 - 08:03 PM

Hi, I wonder is PCR efficiency important in real time absolute quantification? Is is acceptable if the PCR efficiency not in 90-100% but the R2= 0.998? Thanks in advance.

Cheers!
appleyun

#2 warsel

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Posted 18 June 2009 - 04:16 AM

Depends on the method - do you run your housekeeping gene in the same reaction or in another one?
If you run it in another one, then stochastic variations in PCR efficiency will probably affect your result.

Having said that, your Rē looks pretty good to me so I probably wouldn't worry all too much about that.

Running the same positive control in all your PCRs will give you a feel of the day-to-day random variations in your mastermix and will allow you to correct for some of that variation as well.

#3 ivanbio

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Posted 18 June 2009 - 07:46 AM

Hi appleyun,

Your assay efficiency is a measure of how well your amplification is working. The reason why we typically say that an acceptable efficiency should be between 90 and 110% (not that it is 110 not 100%) is because once you start deviating more than that the values you get from your PCR are significantly different than what is real. Think of it this way: is your PCR assay was 100% efficient, then every cycle of PCR will double the amount of DNA present in your reaction. Now, if your efficiency is only 90%, then the true amplification of your DNA is significantly less.

In theory for absolute quantification this is not a big issue since the lower amplification efficiency of your standard curve will be reflected on your unknown samples, so everything evens out. Yet, a lower efficiency also means that your error will be higher and therefore your results not as reliable. A very good R2, like you have, is a good thing, but that is a measure of precision, not accuracy. In other words, your results are very precise, but not accurate.

In summary, I suggest you try to get your assay within 90 to 110% efficiency if at all possible.

Ivan
Carlsbad, CA

#4 appleyun

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Posted 19 June 2009 - 02:06 AM

Depends on the method - do you run your housekeeping gene in the same reaction or in another one?
If you run it in another one, then stochastic variations in PCR efficiency will probably affect your result.

Having said that, your Rē looks pretty good to me so I probably wouldn't worry all too much about that.

Running the same positive control in all your PCRs will give you a feel of the day-to-day random variations in your mastermix and will allow you to correct for some of that variation as well.


Hi! Good day!Thanks for your comments. Will check it out!

#5 appleyun

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Posted 19 June 2009 - 02:33 AM

Hi appleyun,

Your assay efficiency is a measure of how well your amplification is working. The reason why we typically say that an acceptable efficiency should be between 90 and 110% (not that it is 110 not 100%) is because once you start deviating more than that the values you get from your PCR are significantly different than what is real. Think of it this way: is your PCR assay was 100% efficient, then every cycle of PCR will double the amount of DNA present in your reaction. Now, if your efficiency is only 90%, then the true amplification of your DNA is significantly less.

In theory for absolute quantification this is not a big issue since the lower amplification efficiency of your standard curve will be reflected on your unknown samples, so everything evens out. Yet, a lower efficiency also means that your error will be higher and therefore your results not as reliable. A very good R2, like you have, is a good thing, but that is a measure of precision, not accuracy. In other words, your results are very precise, but not accurate.

In summary, I suggest you try to get your assay within 90 to 110% efficiency if at all possible.


Hi,

Thanks ivanbio!
Good day!
Thanks for your comments! I really appreciate it very much. I tried the amplification again and the R2=0.9989 but the PCR efficiency is about 83%. I read about setting baseline and threshold. I then tried to re-set the baseline (rather than following the default setting) but do not make any changes in the threshold setting. The PCR efficiency increases to 91% with R2=0.998. So, my worries now is that whether the data is valid and accurate if I do so?
Any comments are greatly welcomed and appreciated.
Thanks in advance.




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