Spliced and unspliced form of a gene - (Apr/24/2012 )
I am trying to study the spliced and unspliced form of a gene using taqman system. So that means I have two primers and probe for each form (Spliced and unspliced). I already optimized the conditions using RT-PCR for primers and they are working fine.
For loading control I can use GAPDH which is fine. As a control what I have to use for to test the spliced and unspliced genes because different probe/primers for spliced and unspliced have different chemistry and whether those results are because of probe difference or actually spliced/unsplied form.
Can anyone suggest something valuable on this problem?
Thank you for your valuable time and input on it in advance.
Using GAPDH to control for RNA loading is enough, I don't think additional controls are needed because both spliced and unspliced forms are transcribed RNA.
As you have different primer/probe sets for both variants you can't directly compare the normalised Ct values. But you don't need it of you just want to do a ratio of spliced/unspliced variant.You don't even need GAPDH for that (but can keep it for quality control).
If you want to do a comparison between several samples, and you're not looking at the ratio of spliced/unspliced but rather changes in their overal quantity, you would need something to normalise to. GAPDH may be OK, but that depends if it is stable within your conditions. Other stable reference gene may be needed otherwise.
But it's no use to go into details, as you didn't specify what exactly are you going to test and what for.
Also, generaly, it's good to to specificity tests if possible, check that your assays don't bind the other splice variant or DNA if you didn't do it yet.
Hi PCRMAN and TROF,
Thank you both for your valuable suggestions. Indeed these suggestions will be very helpful. Hi Trof, I have one more questions to you and it is that can I use the probe used for spliced in combination with the primers for unspliced because some people recommend to use the same probe. Do, you think it is just sill idea or it may be of worth?