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How to measure Isoforms Expression in tissues?
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  • Robert KibajaRobert Kibaja
Good day everyone,
I work on a protein that can exist as four different isoforms/types. These isoforms arise due to alternative splicing at the juxtamembrane region where you can get either A or B and then at the cytoplasmic region of the same protein/DNA where you can get 1 or 2. Refer to the diagram to see the schematic of my protein. So in essence, because of these combinations, you can get four isoforms A1 (if you get A at juxtamembrane and 1 at cytoplasmic region), A2 (A at juxtamembrane and 2 at cytoplasmic), B1 (B at juxtamembrane and 1 at cytoplasmic) and B2 (B at juxtamembrane and 2 at cytoplasmic).
I am trying to measure the total amounts of these isoforms using qPCR but it's hard because qPCR only allows me to probe for one region at a time, so just juxtamembrane or cytoplasmic region. To get around that, we had the idea of reverse transcribing RNA using the primers for probing 1 at the cytoplasmic region and the primers for probing 2 at the cytoplasmic region. So right now I have some cDNA that only contains DNA/genes with the sequence for 1 at the cytoplasmic region and I have separate cDNA that only contains the sequence for 2 at the cytoplasmic region. So in each of these cDNA, I am trying to figure how much is A and how much is B at the juxtamembrane region using qPCR primers for A and B. I have done that and I got some CT values from the qPCR I ran. Since I used specific primers to reverse transcribe my RNA to cDNA, I do not have any other genes e.g. housekeeping genes for me to compare or normalise the CT values back to. Does anyone know what I should be normalising my CT values from the qPCR? Is there another way of analysing these kind of data? Any help/suggestions welcome and much appreciated.
Thank you very much,
Robert.

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