I have checked mRNA expression pattern of certain genes using partial sequence. I have communicated a paper with the expression result. Reviewer has asked, why have I taken partial sequence instead of full length sequence?
So, I wanted to know whether is it necessary to have full length sequence of mRNA for Real-time PCR?
Thanks in advance.
Dear Saurav. No. In essence not having the full length sequence should not be a barrier to valid assay providing you can demonstrate by BLAST search that your probes are indeed specific. That said with GenBank and Ensembl full length sequences are readily accessible. Just make sure that if you are using multiple probe sets to the same gene that your probe sets are detecting the same isoforms of your gene if this is Relevant to your analysis
once again a simple blast of your primer sequences will take you to the full length ref sequence in Genbank and it will be relatively straightforward therefore to map your probe sets to this gene sequence
If you want a detailed SOP describing exactly how to do this - assuming this relates to a potential concern of the reviewer - I can supply when I return to work on Monday
Real-time PCR always amplify short fragments even shorter than the conventional PCR. For sure the reviewer is asking for another reason and you need to give more details about your study to be able to understand what is the meaning beyond asking this question.
I agree totally with Laurence, you have to be sure that your primers/probes are specific of that sequence but you don't need to know the full-length mRNA sequence. In fact, if you want to see if your primers amplify one product or more than one you can analize melting results in order to see only one product or more than one. Probably it's a conclusive analysis to see that your primers only amplify one product.
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