Hi,
I have treated breast cancer cell line (MCF7) with miRNA mimic. I want to calculate the fold change of miRNA expression between untreated and treated cells. However, the Ct value of miRNA in untreated sample is "undetermined" using real-time qPCR because maybe the miRNA is too low in MCF7. I only have the Ct value of miRNA after treatment with mimic.
How do I calculate the fold change between untreated and treated?
You can determine it in the context of a steady-state control RNA. If it's not expressed at all in the untreated samples, then you will have to show relative expression compared to a control.
You can use more RNA or cDNA in your experiments so that you can prepone Ct value of miRNA in untreated cells. Also, you can increase the number of cycles of your qPCR.
If you do not use Taqman probes, I strongly recommend to use it instead of SYBR green method. The accuracy and sensitivity of probes are much greater than SYBR.
Hello Fahran
I advice you do a pre-amplification in yours experiments or more RNA or cDNA. But in spite of this in your untreated cells the miR is not expressed, you should check some references that support that this miRs is expressed in this type of cell.
Best regards
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