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How to present and analyze these real-time PCR data?


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#1 VirusGuy

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Posted 22 December 2010 - 05:24 PM

Hi all,

I have completed a set of real-time experiments but am unsure how to present the data and perform statistical analysis. I am looking at transcript levels of a certain gene in two different cell lines. I used equivalent amounts of total RNA as input for RT, and then directly performed qPCR with the resulting products. Here is the data:

Cell line #1
Experiment 1: Mean Ct of 3 PCR replicates - 26.5
Experiment 1: Mean Ct of 3 PCR replicates - 26.0
Experiment 1: Mean Ct of 3 PCR replicates - 25.6

Cell line #2
Experiment 1: Mean Ct of 3 PCR replicates - 30.0
Experiment 1: Mean Ct of 3 PCR replicates - 30.0
Experiment 1: Mean Ct of 3 PCR replicates - 30.6

Should I present the data by making cell line #1 100% and then calculating the percentage found in cell line #2? If I do this, what statistical test do I use to determine significance and how do I present SEM? Is a T test sufficient? Can I analyze the Ct values directly?

Any help would be greatly appreciated. Thanks.

#2 VirusGuy

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Posted 22 December 2010 - 05:27 PM

Sorry - just noticed an error on the original post. Should be Experiment 1, 2, and 3 for both cell lines.

#3 qPCRman

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Posted 12 January 2011 - 07:05 AM

Hey

You need to normalize the Cts to the endogenous control- get the DCt and then compare the control to the target group for fold change.

Take a look at this PCR whitepaper http://www.integromi...qPCR_Statistics

and also the Livak paper.

Sam




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