Dear everyone,
I'm working on a project to detect the deletion of exons. We are working with Realtime PCR and using Livak's method. But we don't have a positive control of the target gene, we only have a positive control of a different gene. They are on the same chromosome. Can I use gen A's exon for a positive control to detect a deletion of gen B's exon via Real-time PCR?
We think yes because they follow the same principles. But I don't see any other published paper that did the same way we do. If anyone who has done this before or knows that it's ok or not, please let me know.
Thank you in advance and hope you all have a nice day.
Real time PCR has a lot of problems to tell 1 copy from 2 copies, however well you set your reaction up. Which means that you will have such an amount of uncertainty that you will not be sure whether there is a deletion or not. If you want to test a deletion of exons by a PCR method, the best solution is using MLPA (multiplex ligation probe amplification). Check the MRC Holland website because they may have an assay set up for your particular gene.
Dear Mr Francisco J. Del Castillo,
Thank you for your answer.
We know about MLPA method for detection a deletion of exons but we want to do it by Realtime PCR since it's much cheaper than MLPA and our fundings aren't wealthy. This Realtime PCR method could also be used widely and cheaply if it works too so that is another reason why we want to do Realtime PCR.
Thank you for your suggestion. We may use MLPA result as an positive control to compare with the Realtime PCR result to see weather it works or not.
Thank you and best regards.
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