I am designing primers for my study. I couldn't find answer to my question anywhere.
I will try to see the effects of a drug on transcript levels of different genes.
I intent to cover all mRNA transcripts so that i won't miss anything for the moment and my study in current state, doesn't search for transcript variants.
I am using Blast's "primers for a common group of sequences." and it does wonders.
But i can't be sure about these predicted transcripts and for different genes i encounter different hardships for group specific primers. So my question is, are these
predicted (Xm_) transcripts:
- Necessary to cover all the possible gene transcription,
- Doesn't matter too much to include or not,
- Or should be avoided at all costs?
(A bonus question: Some small transcripts like, Agtr1a, has two exons.
Blast can't design a primer with optimal length, Tm, GC etc. features when i ask for an exon junction or intron spanning primer. I wonder what is my best option here
- Design a bad length, bad Tm, bad GC primer for the sake of Genomic purity,
- or a proper length, Tm and GC but "dirty" primer and leave the job to DNAase?)
I'm sory if my terminology is a bit raw. I am a physiologist and it is my first try with primer design and PCR.
Have you considered RNA sequencing like RNASeq? If you have a large number of genes you want to monitor it might be more cost effective to just do the entire transcriptome.
I work with human cell lines and often find that genes I'm studying are expressed as one of the predicted "XM" transcripts, so I would definitely include them in your primer design. However human genes have lots of isoforms, so for your specific organism maybe XM transcripts are less likely to be expressed.
For your second question, I would choose to design a good primer that doesn't cross an exon-exon junction. DNase treatment is usually pretty robust, but more importantly you should be including some "no RT" controls in your qPCRs, which will tell you if you get amplification from DNA.
Ciaran Daly thanks for your answer. I think it is like what i have guessed then. I will be working with naive Wistar Rats. So probably they won't be too common to have XM transcripts. But it will be good to include them, and it won't definitely be bad to incldue them. So my priority will be including them, as i already did, and with some genes it was hard to capture all, so i included what i could capture with them.
And i will go with the good primer option too, it makes sense, thanks for your answers. It was all i needed.
Robert Adolf Brinzer thanks for your answer also. In my current budget with my dissertation, RNASeq is not cost effective, and my budget and mind is already fixed for PCR.
- Badges
- Science method