Doing 5'-3' RACE and oligocapping combined with sequencing, I can see the primary transcriptional start site for an mRNA change from position 2 to position 30 to position 205 on the same mRNA depending on the way the cells were treated. As this was a single biological replicate I would like to confirm this by qPCR. Is there any reason it won't work to just have a forward primer between position 2 and 30 to study the longest variant and have other forward primers between position 30 and 205 or after 205 to study 2 or 3 variants, respectively?
Mark, I have one doubt with your primers/technique, the longest variant will have binding site for all the three forward primers you mentioned. So all the three sets will amplify longer transcript along with the shorter ones. Hence you won't be able to distinguish between the expression levels of these three. Sorry if I am incorrect. If they are protein coding, you can use splice variant specific antibodies to quantify.
Mark, If all the gene transcripts produced are of the same splice variant in each treatment group, you can use your primers. If the sample is a mixture of more than one variant, I am not sure..
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