In a sequence stretch of human genome, I have found multiple splice donor and splice acceptor sites using software human splice finder. I have to clone a region which precisely produces my desired fragment post expression in target cells ( i.e after expression they splice and express the desired sequence in my target cell) . Can anyone suggest me a method to determine which splice site is preferentially used in cell?
You can do real-time RT-PCR to evaluate the expressed mRNA sequences in your cells of interest. You design primers specific for the different splice variants and then detect their relative expression.
Be sure to select (if you are analyzing endogenous gDNA) or transfect (if you are analyzing exogenous gDNA) an appropriate normalizing gene/construct so that the results from different PCR reactions can be compared.
Hope this helps, good luck!
Becca
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