1 reply to this topic

[cm13]

I'm trying to characterise a protein at the moment.
To do this i have to use real-time PCR.

However, while i have the protocol for getting from cells to real time pcr, i have to design the primers.

And i have no clue how to approach this.
I would use something like pubmed to check the primer design, however the protein im characterising has 3 or 4 different isoforms, and i dont know how to design primers specific to a certain isoform.

Could anyone give me advice on how to do this?

[Trof]

First get mRNA sequences for your izoforms on Entrez or Ensembl (I prefer Ensembl, it better illustrates different transcripts). Check what exons are shared and which are specific. Then design primers with primer3 on the exon-exon boundaries specific for certain isoform, using the target "[]" symbols for the junction. Then blast your primers to see if they are specific.

Another option is to use Universal Probe Library Assay Design Center (it's primary for designing a UPL probe assay, but usualy you can use the primers for SYBR green without a probe) and let the aplication design a differentiation assay for selected isoforms.