Has anyone performed a quantification of multiple splicing variants of the same gene? The gene I'm studying has 7 isoforms and I intend to quantify these from leukocyte sub-populations after FACS sorting (I realize that sampling volume is also a big issue here). Any clues?
Hi,
you might be interested in these papers :-)
http://www.ncbi.nlm.nih.gov/pubmed/11433044
http://www.ncbi.nlm.nih.gov/pubmed/18817741
http://www.ncbi.nlm.nih.gov/pubmed/?term=lincz+mudge
Hi,
If you know the sequence and the "structure" (intro/exons) of your 7 isoforms, you can try to design a couple of specific primers for each of your different isoforms.
Hi,
I would recomand to design primers overlaping two exons to be sure of wich isoform you amplify.
Because you didn't provide the exact gene structure, it is hard to give detailed answers. Generally, if any of your isoforms contains a unique exon, the best way is to design one of the primers on this exon (e.g. BDNF isoforms, see my papers). Otherwise, if any of the isoforms has a unique exon-exon junction, you may have to consider the primers on the span, which gives really limited choices.
Hopefully this helps.
Sometimes conventional (gel-visualized) PCR is the only way to go if your sequences are not amenable to qPCR primer design discrimination between the isoforms. A single primer set designed to the 5' and 3' UTR will give you a nice gel picture of all isoforms present in the sample. Sometimes, mismatches of only 1 or 2 bases in primers for qPCR will still give you amplification of the undesired isoforms. I learned this with VEGF the hard way... at least 7 isoforms involved.
Some qPCR primer designs are simply not possible, even though you absolutely wish them to be. Interrogate your sequences patiently (did I say patiently?) and see if you have other options for primer design.
Hi,
you might be interested in these papers :-)
http://www.ncbi.nlm.nih.gov/pubmed/11433044
http://www.ncbi.nlm.nih.gov/pubmed/18817741
http://www.ncbi.nlm.nih.gov/pubmed/?term=lincz+mudge
In addition to primer design considerations given above, you'll have to choose your reverse transcription method so as not to shift relative representation of your isoforms. i.e. If performing a 2-step RT-qPCR, use random hexamers to prime the RT rather than a poly-dT oligo.
If possible, it may be preferable to use a 1-step RT-qPCR, which also has the benefit of requiring less RNA.
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