8 replies to this topic

[Ensyeh]

Hello every one,
Im beginner in q-PCR .I had to repeat several experiments because they were unexpectable based on samples sites, but I've got some results and I have a question: the CT value is appx the same but the quantity is many different and I dont know why is happend this big difference and how to decide for the true ones? .
need your help

Edited by Ensyeh, 06 March 2013 - 12:56 AM.

[doxorubicin]

I assume you're using the delta-delta Ct method and calculating relative quantity (RQ).  For the delta-delta Ct method you are essentially calculating the ratio of two genes, so your measured gene might give the same Ct for two samples, but the reference gene (GAPDH, 18S, etc.) might have given a different Ct, leading to a different ratio....hence different RQs.  The only problem is that you really don't know if your gene of interest is changing or your reference gene is changing.  Since you are a beginner, it is usually best to assume that most of the big differences can be classified as "noise" associated with technical problems.  Repeat the experiment until you can generate similar results 3 times.  When you publish your data, someone should be able to follow your exact protocol and generate similar data.  If you randomly chose one result over another because it supports your hypothesis, then you're essentially just making up data.

[Ensyeh]

Thank you Veteran , I just want to measure the quantity of my gene of interest in environmental samples using the standard gene with the clear concentration(quantity). in the same quantity of my standard Ive got the same CT and my standard curve is fine . but for my sample it didnt happened and I have the same Ct mean with a big difference in the quantity Mean. (second experiment is showed by red).

Run1:Sample1:CT=31.44781/ CT Mean=31.40128/ CT Standard Deviation=0.065814/ Quantity=1.7*10^-7/ Quantity Mean=1.8*10^-7/QSD=8.5*10^-9/sample 1:CT=31.35474/CT Mean=31.40128/CT SD=0.065814/Quantity=1.9*10^-7/Quantity Mean=1.8*10^-7/QSD=8.5*10^-9/

Run2:sample1:CT= 31.87543/CT Mean=31.46424/CT SD=0.5815/Quantity=1.5*10^-8/Quantity Mean=2.3*10^-8/QSD=1.1*10^-8/Sample1:CT=31.05305/ CT Mean= 31.46424/CT SD=0.5815/Quantity=3*10^8/Quantity Mean= 2.3*10^-8/QSD=1.1*10^-8

Edited by Ensyeh, 06 March 2013 - 02:21 AM.

[doxorubicin]

So the standards gave exactly the same Ct values from Run1 to Run2, so the best fit lines are equivalent?  Your Ct values are not equal actually.  They differ by 0.06.  If everything you say is true, then this difference accounts for the 0.5*10-8 difference in the two values.  What is the calculated efficiency of your standard curve?

[Ensyeh]

Run1: slope-/3.1 /efficiency: 1o9 ,Run2: slope:-2.5 / efficiency:145 Run1: CTmean for 0.2 ng standard:12.3 Run2:CTmean for 0.2 ng standard:12.4. My supervisor believes that my standard is the problem, since although the amount of plasmid DNA are the same (based on photometry) but copy number of target gene are not the same.

Edited by Ensyeh, 06 March 2013 - 04:12 AM.

[doxorubicin]

So your standard curves are quite different actually.  For the 0.2ng standard you might have similar Cts (12.4), but the slope is quite different, so the other Cts for the other dilutions on the standard curve are different.  This explains why the same Ct (31.4) gives a very different quantity from run-to-run.  Both efficiencies you have (but especially the 145% efficiency) are theoretically impossible....100% means your replicon doubles each round of PCR.

Are you making the standard curve each time?  I make a single standard curve diluted in TE buffer with sheared salmon sperm DNA for stability and use the same standards for every run.

[Ensyeh]

Why I didn't think to this great option myself? thank you much! (I was making the standard dilution each time). I should try it. thanks again.

[Ensyeh]

Let me to ask my last question : how many times you are using the single standard dilution? and if you keep your standard in -70°C or -20?
thanks a lot.

[doxorubicin]

I keep them at -20C and use them for years.  They are very stable.