does anybody know, if it feasible is, to try finding splicing variants with standard PCR with cDNA made from total mRNA? I am trying to design primers for a standard PCR starting before the first possible lacking exon and ending after the last lacking exon, and check it on Agarose Gel if any difference in size occurs. But the Primers do not work. In other papers the splicing variants are investigated either through cloning and sequencing or through ratio between different isoforms in Real-time PCR. Does anyone have an idea if it would be possible to see the absolute presence of isoforms like this?and if yes (in prizip it should be possible) of what should I consider?
Best Regards
Hi,
You can use primerblast to make primers that target specific exons using RT-qPCR (non-gel method).
Please read this as if shows examples of exon/transcript specific primers:
http://nar.oxfordjournals.org/content/early/2016/04/19/nar.gkw263.full.pdf+html
Asif
Dear Tannaz
- Yes it definitely is possible and have done it recently for a gene isoform recently
- What is more in the absence of published literature it will tell you which splice form is dominant/exclusive
- A few years back I was part of a functional genomics initiative and my role was exomic sequencing of all genes within a GWAS loci. As you can imagine that involved making hundreds of primers to many genes and splice variants therein
- perhaps 30% of such primers didn't work and invariably this was due to transcript models being wrong
- Thus I agree with Asif: use primer blast to design isoform specific primers but check your RefSeq prediction against ENSEMBL (EBI) as well
- I am now in the habit of just designing isoform specific primers to exons where both data bases agree
- Take into account as well that isoforms are nearly always tissue specific so look for empirical evidence of what is expressed where in the literature
- Furthermore even with correct predictions some primers do not work so I tend to design at least 2 primer pairs to my region of interest
- Finally look at your target sequence as well: some primers are inherently sound but their target sequence is either more than 60% GC rich or consists of regions with more than 4 G/C residues together both of which reduce PCR amplification efficiency
- The same type of motifs but AT rich are if anything even more difficult to amplify efficiently
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