Statistics: real-time RT-PCR - (Nov/17/2007 )
Hello!
I have following scenario:
Two groups - treated and non-treated patients:
I have 3 gens and 1 housekeeping gene (hkg).
Real-time PCR with 40 samples and 20 control samples (ex vivo cells)
I got delta ct´s from all samples with regards to the hkg´s.
Can I use delta delta ct methods for calculation of the gene expression ratio? or shall I simply use a t-test for the deltas?
Can I express all deltas as median or means and standard deviation and put the values in the dd-ct equation?
Thanks for help!
Well, I can't really answer your question, but in the lab I am staying they now use software (REST 2005) for statistical evaluation of PCRs. By now I haven't reached the point where I needed to test this, so I'm not sure if this is of any help!
First hit in google when searching for REST 2005!
I agree that data management software is your friend. this is from Biotechniques' site - it used to be free but you'll have to check?
qGENE
Q-Gene manages and expedites the planning, performance, and evaluation of quantitative real-time PCR experiments, as well as the mathematical and statistical analysis, storage, and graphical presentation of the data. The Q-Gene software application is a tool to cope with complex quantitative real-time PCR experiments at a high-throughput scale and considerably expedites and rationalizes the experimental setup, data analysis, and data management while ensuring highest reproducibility.
Download qGENE software HERE at BioTechniques
http://www.gene-quantification.de/download.html#qgene
if that's not possible, I would recommend the ddCt method outlined in user bulletin #2 from ABI. a bit of a pain to start with, then works like a charm every time.
Hello,
I have a very similar protocol, and i'm using Genex program to do all my calculations. I don't have to calculate delta Ct's! I directly use Ct values from my target genes and from my endogenous controls. With the genex you will end up with the calculation of the expression ratio, and then you can go for some statistics.
About the t-test or some parametric test be sure that your data have a normal (gaussian) distribution.
Genex is available as a trial www.multid.se
I hope this help.
*R
I have following scenario:
Two groups - treated and non-treated patients:
I have 3 gens and 1 housekeeping gene (hkg).
Real-time PCR with 40 samples and 20 control samples (ex vivo cells)
I got delta ct´s from all samples with regards to the hkg´s.
Can I use delta delta ct methods for calculation of the gene expression ratio? or shall I simply use a t-test for the deltas?
Can I express all deltas as median or means and standard deviation and put the values in the dd-ct equation?
Thanks for help!
Hi Caine,
I am sorry that it has taken me so long to respond to your email. Here is the link to that paper you wanted:
http://www.biomedcentral.com/1471-2105/7/85
Take a look and see what you think. If you have any questions feel free to ask and I will see if I can help. I would use a t-test for your deltas if your data are normally distributed, or if you have a large "n", and if you have a small n, eg n=3, then use a wilcoxon test, which does the same thing as the t-test but for data that are not normally distributed and small sample sizes. Good Luck!