**30**wrote:

Hello,

I'm trying to learn how to analyze miRNA data in R and I'm a bit confused about what the proper way to calculate delta Ct and delta delta CT are.. I have a dataframe with the first column being the miRNA, second column is Task (target/control) and then I have the samples (10 columns: 5 treatment groups and 5 control groups). I've been trying to find examples with data similar to mine for how to compute the delta CT but haven't found anything helpful so far. Could someone please help me by providing a simple example calculation for a dataset similar to mine,for delta CT and delta delta CT? Do I calculate the mean for every column and then subtract the mean from each value in the column to get the delta CT or how does it work?

**Edit:**

I have something that looks like this:

```
miRNA<-c("hsa-miR-10a-4373153", "hsa-miR-10b-4395329", "MammU6-4395470", "MammU6-4395470", "hsa-miR-15a-4373123")
Task<-c("Target", "Target", "Control", "Control", "Target")
C1<-c(28.005966, 30.806433, 17.341375, 17.40666, 30.039436)
T2<-c(30.973469, 29.236025, 30.41161, 20.914383, 20.904331)
C3<-c(26.322796, 25.542833, 22.460772, 19.972183, 30.409641)
T4<-c(26.441898, 25.837685, 23.158352, 20.379173, 33.81327)
C5<-c(39.750206, 19.901133, 28.180124, 22.668673, 25.748884)
T6<-c(23.004385, 28.472675, 23.81621, 26.433413, 28.851719)
T7<-c(22.239546, 28.741674, 23.754929, 26.015385, 28.16368)
T8<-c(29.590443, 30.041988, 21.323061, 24.272501, 18.099016)
C9<-c(15.856442, 22.64224, 29.629637, 25.374926, 22.356894)
C10<-c(38.137985, 24.753338, 26.986668, 24.578161, 19.223558)
data<-data.frame(miRNA, Task, C1, T2, C3, T4, C5, T6, T7, T8, C9, C10)
```

where C1, C3,..C10 are the control samples and T2, T4,...T8 are the treatment samples.

Thanks for formatting the code properly. Next time please either edit your original post or reply to mine but don't add a comments. Comments are for answers.

Question: what is

`Task`

for?edit: I think the

`pcr`

package is a good start for you. It has good documentation: https://cran.r-project.org/web/packages/pcr/vignettes/qpcr_analysis.htmlBut I realise you are asking HOW to calculate deltaCT and this doesn't answer your question. The issue is I'm not seeing your experimental design or rather I'm not understanding your data. If you elaborate we can help you some more.

620Thank you for the suggestions, Amar! I'm trying to understand the data myself since I haven't dealt with miRNA data before. Basically, I think the Task column (Target /Control) represents the target gene vs housekeeping gene? I'm just trying to clarify what is the right way to calculate delta CT and delta delta CT based on this specific dataset I have, since the various posts/articles I've read have honestly just left me even more confused..

30Can you please edit the title of your question? "

Delta CT in R" is very meaningless.Quoting from How To Ask Good Questions On Technical And Scientific Forums

4.6kThank you for the suggestion, I have made the change. Hopefully, the new title is more appropriate.

30Does this help? - A: How to report and plot qPCR data

52kThat's extremely helpful, thank you Kevin! I have asked this question in my reply to dsull as well but just to confirm, if I follow your calculation steps in the link and apply that algorithm to my dataset, I'd basically have 2 delta CTs for each gene, right? One for the C samples and one for the T sample? And then the delta delta CT for that gene would just be the difference between the two deltas, correct? And finally, if let's say I wanted to perform a Limma test to compare the C and T groups, I assume the test would have to be performed using those delta CT values, right?

30Replied to your comment in my answer below.

550