Calculating ct values in qPCR for sample of timepoints to give a relative gene expression
1
0
Entering edit mode
2.1 years ago

I have run a qPCR experiment investigating a variety of genes for a time series of samples and was wondering how to calculate my ΔΔct values in relation to this. I have control genes for each sample (gapdh) which vary slightly for each and I have a variety of genes I am testing the expression of. When calculating ΔΔct values I know you are meant to do it relative to your control gene (ie gapdh) and then relative to your control sample. However as I have a time series I do not have a specific control sample as each gene expression pattern varies from time point to time point. Any ideas how to do this please?

qPCR ΔΔct fold change relative expression • 522 views
ADD COMMENT
0
Entering edit mode

I realize that GAPDH is a prominent (maybe the most prominent) "housekeeping" gene to normalize against. Still, I encourage you to check (if possible) your own or some published related RNA-seq datasets towards expression of that gene. I've seen it repetitively in both published and my own data that GAPDH is significantly up/downregulated between conditions. Your normalization might be massively skewed if using GAPDH. I am pretty sure there is literature out there that investigated GAPDH as a normalization control, though I do not have the articles at hand right now. Might be worth checking. If you have (related) RNA-seq data check for properly-expressed genes with strong evidence (e.g. adjusted p-value > 0.5) against differential expression of that gene.

ADD REPLY
0
Entering edit mode

I have other housekeeping control genes and will be normalizing against those also

ADD REPLY
0
Entering edit mode

That is good. If results confirm each other you probably will be fine.

ADD REPLY
0
Entering edit mode
2.1 years ago
Eslam Samir ▴ 110

Hi,

I think this answer is more than what you will need for your calculations: https://www.researchgate.net/post/How_can_I_Calculate_expression_fold_with_CT_values

ADD COMMENT
0
Entering edit mode

Hi,

Unfortunately that does not make sense for my data as it assumes a control data set (treated vs untreated) whereas my data are just a different points on a time scale

ADD REPLY

Login before adding your answer.

Traffic: 2299 users visited in the last hour
Help About
FAQ
Access RSS
API
Stats

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.

Powered by the version 2.3.6