This is a bizarre problem that happened a couple of years ago to myself and is now happening to a colleague.
The aim is to design primers for PCR using a de novo transcriptome assembly. However, the primers do not seem to work at all (or only a small percentage work at all, eg 1 out of 10). People have been tearing their hair out trying to solve this problem so any input would be greatly appreciated. These are experienced people with years of experience designing primers, which is why this situation is quite so frustrating. We have been going through the possible explanations.
- Is the assembly bad?
The regions upon which the primers are well assembled and have very high coverage. The regions are also highly conserved between species.
- Is there a problem with the cDNA or the RNA?
Different older sets of primers work with the cDNA. Different RNA collections (from the same tissue and developmental time point that was used in the assemby) have been used, and different methods of cDNA synthesis have been used and can be used as template for successulf PCR reactions with older primers.
- Is there a problem with the order/preparation?
The primers were re-ordered and within the re-order, sets of primers that had previously shown to work were included. Again, the old primers worked, but the new ones didn't.
- Is there a problem with primer design?
The primers were designed in Geneious and on the Primer3 webstie (Geneious also uses the Primer3 method).
All the appropriate positive controls have been applied, different PCR mixes have been used. Nothing is working.
Has anyone ever experienced anything like this? If so, would they have any idea why this might be happening? One or two primers not working is normal but entire batches across 10+ genes is very strange indeed.
Thank you in advance for any help that you can provide.
The aim is to design primers for PCR using a de novo transcriptome assembly. However, the primers do not seem to work at all (or only a small percentage work at all, eg 1 out of 10). People have been tearing their hair out trying to solve this problem so any input would be greatly appreciated. These are experienced people with years of experience designing primers, which is why this situation is quite so frustrating. We have been going through the possible explanations.
- Is the assembly bad?
The regions upon which the primers are well assembled and have very high coverage. The regions are also highly conserved between species.
- Is there a problem with the cDNA or the RNA?
Different older sets of primers work with the cDNA. Different RNA collections (from the same tissue and developmental time point that was used in the assemby) have been used, and different methods of cDNA synthesis have been used and can be used as template for successulf PCR reactions with older primers.
- Is there a problem with the order/preparation?
The primers were re-ordered and within the re-order, sets of primers that had previously shown to work were included. Again, the old primers worked, but the new ones didn't.
- Is there a problem with primer design?
The primers were designed in Geneious and on the Primer3 webstie (Geneious also uses the Primer3 method).
All the appropriate positive controls have been applied, different PCR mixes have been used. Nothing is working.
Has anyone ever experienced anything like this? If so, would they have any idea why this might be happening? One or two primers not working is normal but entire batches across 10+ genes is very strange indeed.
Thank you in advance for any help that you can provide.