I just started working with ReadqPCR and NormqPCR packages (Bioconductor) in R. However, I'm stucked 'cause I can't use deltaDeltaCq() function. First things first...
1) I have a group of 8 control patients and 83 diseased patients. I measured the Cq values of ACTB and a gene of interest (GOI) in all of these patients. What I want to do is to apply deltaDeltaCq() function in this cohort data.
2) I used read.qPCR (from ReadqPCR) to read in a table with raw Cq values. It was like this:
Sample Detector Cq
1 control GOI 0.14275905
2 control ACTB -0.75616034
3 disease GOI -0.32048895
4 disease ACTB -0.29002120
5 control GOI 0.51930627
6 control ACTB 0.09161364
...
- I formatted it this way 'cause ReadqPCR vignette says it must have these variables called like that.
3) I tried to use deltaDeltaCq() but without success. It always throw me an error saying I must specify the housekeeping gene I want to use. However, even when I specified it, it said to me that the name didn't exist. I found later that when the qPCRBatch object was generated by the read.qPCR() function, the names of the genes changed being added a suffix "_Tech.Reps(n)", with n being the number of the "technical replicate". My problem is that I don't have technical replicates here. They're all biological replicates.
What am I missing here? Why the qPCRBatch object thinks that my biological replicates are technical replicates?
Thank you.
- Last thoughts:
- I know deltaDeltaCq requires a contrast matrix (contM) telling which ones are data from control and from test groups. However, my data already tells it. How can I proceed from here? I am not willing to build a contrast matrix with almost 100 Cq values by hand.
