quantifying spice variant

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quantifying spice variant

Postby kshanik » Aug 21 2017 1:47 am

Dear All,

I am interested in analyzing a gene that has two splice variants. I know that both of them are expressed - as seen by semi-quantitative RT-PCR. I can design unique primers only against 1 of the two variants. Primers targeting the second variant also amplify the first one (though the amplicon sizes are different).

I want to investigate if there is any difference in their temporal profiles or not. I assume that I can do that by looking at the melt curve data (I should get two peaks since the product size is different – by ~100bp).
But is there any way I can quantify their expression levels?
Another related question – how to quantify changes in their expression levels under different conditions?

I have never worked with genes having multiple splice variants, so I’ll appreciate any help regarding this issue. Thank you very much.
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Re: quantifying spice variant

Postby 29yrsExperience » Sep 12 2017 9:37 am

This is hard to answer without a diagram comparing the two variants. If you can truly only distinguish them by the size of the amplicon(s) in a single tube reaction, quantification will be affected by the relative efficiencies by which the variants are amplified, and as you know the smaller one generally amplifies more efficiently. I don’t think it is “legit” to use the melt curves to quantify the two products. Are you sure you cannot design primers across splice junctions to distinguish them? You can use a common primer at one end (that would stick to both variants) but have unique primers at the other ends. In any case you should also amplify a reference gene like B-actin for normalization.
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