problem with analyzing real time PCR data

[Laleh]

Hi everyone

I have performed an relative quantification on two different samples one cultured cells, one my cells on a tissue engineering scaffold. I have had two replicates in each real time PCR assay, and conducted the experiment two times. while in my control group which only consists of cells the Ct for house keeping gene-GAPDH-is relatively low-around 21- the Ct for GAPDH is around 31 in my test group-cells on scaffold. I have attached the Ct results. I was wondering if this result can be valid or scaffold somehow affects GAPDH expression? please note that the Ct for my gene of interest does not have much difference between test and control. 

Cts.docx

[CharonY]

First, let me apologize for not downloading a document from a first time poster due to security concerns. But I think most of the issues can be diagnosed within a post (or screenshots, if needed). The differences you are seeing are massive (about 1000-fold) so there is good chance that we are not looking at a biological but rather an analytical and/or pre-analytical issue. 

House-keeping genes are not really that universal as they are sometimes claimed to be, but that level of change is extremely unusual. So the most likely scenarios are issues during sample prep and/or the qPCR itself. You mentioned that the ct of your gene of interest remained stable. What is the ct/cq? The next thing to look at is to inspect your curves, do you have stable amplification for all your targets? Are you using probes? If not, you could inspect melting curves.

Also, what is the variance of your results?

[Laleh]

Dear CharonY

Thank you very much for your comments. 

We use HighRox SYBR green mix on ABI stepone plus

I have included Cts here. Please take a look:

 

Cell on scaffold (test): Ct1 for gene of interest: 34, Ct2 for gene of interest: 33. Ct1 for GAPDH: 31, Ct2 for GAPDH: 31

Control cells (without scaffold): Ct1 for gene of interest: 32, Ct2 for gene of interest: 32. Ct1 for GAPDH: 21, Ct2 for GAPDH: 22

 

I do not have access to the melt curves right now

In your opinion what causes the huge difference in Cts of GAPDH between samples?

Thanks a lot

[StringJunky]

I've snipped it here:

[Laleh]

Thank you 

[CharonY]

Ok, so there is at least some hints regarding pipetting errors in the qPCR (a delta of 1 is about a 2-fold change, which should not be happening in replicates). But obviously that does not seem to be an issue with the GAPDH. Obviously, we do not know if it is realistic whether your target is about 1000-fold different in abundance and that the differences are caused by differential extraction (I would consider it somewhat unlikely though).

Main things I would suspect fall into the area of sample handling, and potential assay issues. So a few things to check include:

- quality of mRNA samples

- was it a 2-step? Can there be issues there?

- are the protocols well-established for the specific primer combinations? What are the PCR efficiencies for them?

- is there a possibility of contamination? What levels do you typically have for an extract of your control sample?

- is there a possibility of degradation?

 

[Laleh]

Dear CharonY

Thank you very much for your comments.

I will certainly repeat the experiment and increase the replicates, while checking the quality of RNA and melt curve more carefully. 

Thanks a lot