Hello!
My final objective is to perform ISH. But I’m still thinking a lot about the very first step: primer design.
I have read about the different things to consider when designing regular primers; but, I would like to ask for some clarifications on designing primer for riboprobe synthesis. Starting material: de novo transcriptome assembly
My queries:
A) I am confused with what I have been reading on the net: Should I or I should not design my primers along the identified UTRs of the transcript? If designing on the CDS, should I avoid primers binding on regions with identified protein domains (thru blast) because they might be highly similar to other genes of the same family? Do I need to take this into account wrt my objective?
B) If the gene from the de novo assembly happens to have ‘isoforms’, do I just design the primer in the common region of these Isoforms? Or is it safe to just get the longest representative and design primer for it. Will there be a problem in the ISHif there’s a differential transcript usage?
C) i will use primers with t7 on the reverse pair. Should I design my primers with the T7 promoter in consideration (for the Tm, secondary structure) or just design the primer pair first then manually attaching the T7 to the reverse?
D) I would like to know how successful you are with template amplification (cdna amplified using primers with T7) when you add Spacers such as GG (NEB suggestion) or GAG (one lab protocol I read) or random 5mers in between the T7 seq and Reverse primer.
E) the top most blast hit of my transcript is my gene of interest (eg hox5). However, there are some other genes with significant hits as well (really close eval for hox1, hox2, and hox3, for example). Do I just drop this transcript as it may not be the real gene of interest? I have a lot of these transcripts btw.
Thank you everyone. Keep safe.
