I am trying to design some priers for RT-PCR. As my genes have many isoforms, so I have to design primer for conserved sequences. but the results aren't satisfactory.
There are two ways to tackle this problem. One is to try and design primers for conserved regions and try to amplify all of the possible variants, which you say isn't working as well as your like. The other solution is to design primers to the unique sequences for each varied and amplify them individually. Either way, it's a LOT of trouble-shooting. Alternately, you could use RNAseq and solve the problem bioinformatically. It might be cheaper!
you could design primers include the whole genomic DNA, so that can amplify all the isoforms
Hello Samaneh,
When you are trying to design new primers the more important is the numbers of sequences (datas) aligned from the target gene that you want amplify. If in this alignments you didn't find conservative regions, you can try to design degenerate primers (consider that you can lose sensitivity). In addition, you can design different probes with different fluorophores detecting this "isoforms".
Also, you can use this degenerate primers to obtain more sequences from different samples and try again design new primers more specifics.
In other hand, If you are considering do RNAseq, of course, you can obtain more data for your design, but take into account that you will need to analyse more than one sample, for try obtain all the "isoforms".
If you give more information about the sample that you will analyse, the name of the target gene I can give you more recommendations.
regards
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