qPCR differential gene expression

Question

[ehruan]

Hi, I am validating RNAseq differential expression with qPCR. I have data from qPCR with expression levels from samples from lowest expressed quartile and the highest expressed quartile. I also have 2 normalizer genes. How can I calculate differential expression between highest expressed quartile and lowest expressed quartile with my normalizer gene. Thanks!

Answer 1

[i.sudbery]

The normal method would be the delta-delta ct method. This seems to be a good tutorial: https://2017-spring-bioinfo.readthedocs.io/en/latest/Delta-Delta_Ct_Method.html

[ehruan]

If i have 2 housekeeping genes should I take the geometric mean or just the average of them

[i.sudbery]

You should look at the GeNorm paper/method. https://genomebiology.biomedcentral.com/articles/10.1186/gb-2002-3-7-research0034

[ehruan]

the delta-delta ct method seems to be great for comparing normal vs tumor. I have highest quartile and lowest quartile so when I use the lowest quartile as the control group I get conflicting values. Is there another method that doesn't use this internal control?

[i.sudbery]

What do you mean "conflicting values"? If you don't want to do an internal control, you can just do deltaCt, rather than delta-delta-Ct. (i.e. just 2^(test_gene-housekeeping_gene)