I am trying to perform a RT-qPCR and I would like to design a primerset for one single transcript variant for RT-qPCR. The problem is that it is very difficult to get primers that only bind on one specific transcript variant.
I was wondering if this is possible (I don't think it's possible). But if this is possible, do you have any tips? I have to perform gene expression analysis for one particular isoform and this is my first time.
Hello
You should do a multi-alignment of all transcripts then find the region in your transcript which is unique and have no similarity with other transcript. Based on your findings you have 2 options:
1-You may put both of primers in a region unique to your transcript
2-You may only put one of the primers in that unique region and the other one in a common region
It will give you a specific fragment for your transcript.
Sure, you must find the differences in the sequence (alternative exon/sequence) which is different to the other splice variants (e.g. via using the "Multialin" tool). For this sequence you can finaly prepare a (For or Rev) primer (which is only present in this sequence passage). The other For or Rev primer can be the same as found in all splice variants. Thereby, you should be able to detect the spice variant you are interested in by RT-PCR.
The advantage of performing quantitative RT-PCR for expression analysis is that it does not need the primers that cover the whole sequence of genes or splice variants. So, in your case, you can find the stretch of sequence, which has some differences from other splice variants. This can be further chosen to design the primers.
Hello
You should do a multi-alignment of all transcripts then find the region in your transcript which is unique and have no similarity with other transcript. Based on your findings you have 2 options:
1-You may put both of primers in a region unique to your transcript
2-You may only put one of the primers in that unique region and the other one in a common region
It will give you a specific fragment for your transcript.
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