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I'm working on a mock research proposal for an undergrad molecular techniques course, and having a bit of trouble getting my ideas straight.
The basis of the proposal is that an assay should be designed to detect and measure the relative amounts of two alternatively spliced transcripts in the muscle tissue of mice (at different time points, i.e. young, adolescent, adult, etc.) The only starting material given is a portion of the original gene cloned into a pBluescript vector, like so:
-----| NsiI | Exon 1 | Intron | Exon 2 | Intron | Exon 3 | Intron-ClaI-Intron | Exon 4 | SmaI |--------
The two transcripts of interest are gene1,2,3,4 and gene1,2,4 (i.e. the second transcript does not contain exon 3). As depicted above, the original gene has been cloned in between the NsiI and SmaI sites of the vector. There is also a ClaI site in the intron between exons 3 and 4. Additionally, the NsiI site occurs in the middle of exon 1 in the original gene, meaning the leading portion of exon 1 has not been cloned into the vector.
I'm thinking the best way to measure the abundance of the two transcripts in each tissue would be to design forward primers for the unique splice junction sites of each (i.e. 2/3 for the first, and 2/4 for the second) and do non-competitive RT-PCR. However, I am a bit stuck on how to design the primers since I don't have the actual sequence for the gene, and even though I know it contains the four exons etc. I don't actually know where they start and end.
So then I was thinking I could do a Northern blot to detect the presence of the two transcripts, then isolate the transcripts from the original gel and have those sequenced to determine where they differ (i.e. at first point of difference that would be the splice junction site differences?). Although I am a bit confused about how to create a probe for the Northern as well.. I was thinking maybe I could excise exon 4 from the vector using the ClaI/SmaI restriction sites and label that?
I haven't had a chance to talk to my professor yet, but I also don't want to look like a total dumbass when I go to discuss things with him lol.. One of my classmates mentioned that he thought the best technique to use for the assay would be an RNAse protection assay, however I'm not sure how I could design probes specific for each transcript (similar to how I can't design primers for RT-PCR! So confused).
Any help or assistance is greatly appreciated!
