I am having a hard time doing custom taqman RT-PCR genotyping because I designed my own primers and probes. I can say the protocol is working, but I am getting very low fluorescence intensity for my probes. Is it because I am using cDNA? is genotyping via RT-PCR should be using gDNA? Can you share your protocols using this technique. Thank you so much.
First of all, what is your experimental setup, what are you genotyping?
And second, if your gene of interest is expressed only at low levels using RT-PCR you will of course only obtain low fluorescence intensities. In addition, if your gene shows allele-specific differences in expression, RT-PCR will confound your results. I would rather design a gDNA-based genotyping assay.
Yes, you can use cDNA for genotyping using RT-PCR. However, the choice of a template (cDNA or gDNA) depends on the specific application and the type of genotyping you are performing. For example, if you are interested in detecting somatic mutations or copy number variations, you may want to use gDNA as the template. On the other hand, if you are interested in gene expression analysis or detecting germline mutations, cDNA may be a better choice.
Regarding your low fluorescence intensity issue, there could be several factors that are contributing to this problem. One possible explanation is that the design of your primers and probes may not be optimal for the specific target sequence you are trying to amplify. You may want to consider using a different design algorithm or consulting with a bioinformatics expert to optimize your primer and probe design.
Another possible explanation is that your cDNA template may not be of high enough quality or quantity. You may want to consider optimizing your RNA isolation and cDNA synthesis protocols to ensure that you are obtaining high-quality cDNA.
Thank you
Khalid Khan
Why would cDNA be the better choice for detecting germline mutations? In my opinion gDNA should be used.
Sabine Strehl
cDNA is the better choice for detecting germline mutations because it represents the expressed genes in the cell, whereas gDNA represents the entire genome. Germline mutations are heritable changes that are present in every cell of an individual's body, including both the DNA that is transcribed into RNA and the DNA that is not transcribed. Therefore, cDNA provides a more accurate representation of the mutations that are actually being expressed in the cell. In contrast, gDNA may contain mutations that are not expressed and may not be relevant to the phenotype under investigation.
This is textbook knowledge and certainly right, but in case of splice site mutations or relevant mutations in non-coding regions the only option is using gDNA.
Thank you for your answers. I think I have to go with gDNA this time, since I've tried a couple of experiments with cDNA. It worked with my "synthesized gene" as control, but not very with my cDNA samples. I am working on animal samples. I'm good with my gene expression using the RNA extracted and cDNA, so there's not much of a problem wtih my quality and quantity. I'll move to gDNA as my next step. Thank you very much and I appreciate your help.
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