Some techniques involve averaging exon expression levels, but this can be a problem for genes that exhibit alternative splicing, so I'm wondering if there's a more stable region to take to get a more accurate idea for total gene expression. Maybe the 3'UTR?
Sharmin Siddique Bhuiyan
I usually take half coding sequence of the last exon (~250 bp) and half of the 3'UTR (250~350bp) and design a primer for about 500~600 bp. It worked great for me all the time to check the gene expression. Best wishes!
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Laurence Stuart Dawkins-Hall
- I completely agree with the last comment
- The other reason for choosing the last coding exon and/or 3 UTR is that reverse transcriptions from the 3' to 5' so representation of the terminal 1-2kb is always maximal
- In addition RNA degradation proceeds from 5' to 3' so also from the point of view of RNA stability the last 1kb of a transcript is best
- Finally if you are interested in assaying total expression irrespective of spliceform variants then situating your primers within the 3' UTR is most effective
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