I want to compare gene expression between 3 patients and 3 controls to investigate if there is any difference between the two groups. I've ran a RT-qPCR using SYBR Green. Should I use RQ values? Which test should I apply?
My colleagues use Student's T-test. but I'm not sure whether I can use it for a small samples (n=3).
Thanks in advance! :)
Concerning small sample size: 3 replicates are far from ideal but a two sample t-Test is probably the "safest" that you can perform. The consequences of such small sample sizes for nonparametric tests are worse (e.g. U-Test or npar.t.est).
Talking about t-Test, since it is hard to assume anything on the variances with 3 replicates, you might want to do the Welch t-Test (that is the default in many Statistics software packages for t-Test).
Yes, it's resonable to use a t-test, but not on RQ values. The test should an independent-sample t-test on the dCt values.*
*Assuming RQ is something like 2^(-)ddCt. Background: dCt-values are pretty well normal distributed, and the transformation will distort that nice property. The t-test assumes normal distributed data, what is ok for dCt values. As you have independent samples, it's best to use an independent-sample t-test rather then to calculate a mean ddCt along with it's SE to then try to get the t-statistic manually for that single value and then find the corresponding p-value, considering the correct degrees-of-freedom.... It's far simpler to just use two independent groups (patients, controls) of the dCt values - any standard software will do a t-test with such data.
Hello Sofia, I would have to agree with my colleagues. The sample size is not important for t-test as long as the data is drawn from a normally distributed population.
I agree, a t-test is possible for normally distributed samples. Additionally nonparametric tests are possible for dependently distributed samples.
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