Real time pcr -sensitivity ?

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Diablo
Diablo's picture
Real time pcr -sensitivity ?

Hi guys,

I study biotechnology and i did my first RT-PCR last week. So we made a standardcurve (dilution 10^7 till 10^0), measured our samples and printed the report.
Then there was a question about the sensitivity of our experiment. As far as i understood the sensitivity is that ct-value of the slope, where the slope hits the y-intercept (at 10^0 = 1 molecule) --> is that right ?

heres an pic of the quantitation info --> so the sensitivity would be about 34 ct ?

[img=http://img01.picoodle.com/img/img01/7/2/5/t_quantiatii_7aa7m_795502d.jpg]

Greets Diablo

Tony Rook
Tony Rook's picture
Hi Diablo:

Hi Diablo:

I believe what you are looking for is how to determine the amplification efficiency of your real time PCR reaction. This is really what is important when doing real time PCR. Gene Quantification is a great resource for all things concerning quantitative PCR.

Here is a link to their PCR Efficiency page which contains a bunch of resources and presentations talking about amplification efficiency and what it means.

If you find this information helpful please let Scientist Solutions know by posting a success stories at:
http://www.scientistsolutions.com/t1262-Success+Stories.html

Diablo
Diablo's picture
Thx for your info !

Thx for your info !

Yeah the efficiency was also a question --> i just took the value from the report (the programm calculated it automatic, but your infosite is helpfull for explanation)
--> But actually i dont think its the same : cause there are
some question and one is:
What is the efficiency of the RT-PCR analysis with GUS primer
(the experiment we did was with GUS reportergen)
and another question:
What is the sensitivity of ur RT-PCR analyses (we did 2 )

And the only info in the script we were given is: "The y-intercept correlates with the sensitivity of the real -time reaction and the Ct-value of the blind control"
--> is this efficiency ?
(there is a picture of a standard slope which hits the y-intercept at 10^0 Molecules..)

So that's why im a bit confused.... i googled the internet and yes they are all talking about efficiency but actually i dont find something about sensitivity...

Greets Diabloooo

Tony Rook
Tony Rook's picture
Diablo:

Diablo:

Let me review the steps involved in constructing a standard curve and the pieces of information generated from this curve. Standard curves in quantitative real time PCR are often utilized to quantitate the number of copies of specific gene from your sample.

Step 1. A series of 5 to 6 serial dilutions (2, 5, or 10-fold dilutions may be used) of your standard should be prepared. The concentration range should match the anticipated concentration of the sample to be analyzed.

Step 2. Serial dilutions are analyzed by quantitative real time PCR in seperate wells but within the same run. The resulting Ct of each dilutions are recorded. The sample to be analyzed should also run within the same plate (but different well) as the serial dilution.

Step 3. A plot Ct vs log of copy number should be constructed. This is the standard curve or the linear regression line through your data points. The standard curve can be used to extrapolate the number of copies of your target gene(s).

Several pieces of information can be obtained from the standard curve. For instance Y = -3.43x + 40.4 with an R-squared value of 0.989

R-squared value - The square of the coefficient of regression indicates how well the line fits the data. A quantitative real time PCR linear regression should have an R-squared value of > 0.98.

Efficiency of Amplification - The best possible amplification efficiency would be 100%, or an amplification reaction which doubles the template after each cycle during exponential amplification (that is during the cycles where the original Ct curve plot is exponential). The slope of the standard curve is used to calculate efficiency by the following equation:

Efficiency = [10^(-1/slope)] -1.

The efficiency of amplification in the above standard curve would be 95.7%

y-intercept - The y-intercept is less reproducible than the slop but gives some indication of sensitivity of the assay. Since quantitative real time PCR is very technique sensitive, this number is much more variable. Simple variations in pipetting techniques can effect the y-intercept. The y-intercept predicts how many cycles will be required to be certain that there is no target present in the real time PCR reaction. For this example, this would be 40.4 cycles.

Hope this helps clear some things up,

Tony

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Diablo
Diablo's picture
ok thank you very much !

ok thank you very much !