I am looking for suggestions of methods to design qPCR primers from de novo assembled isotigs in order to further study differential expression of wound-related genes in a non-model grass species.
I have run BLASTX on my assembled contigs/isotigs and now have potential genes of interest that I want to design primers for doing further in-depth qPCR expression analysis on (looking at each gene over several time points post wounding).
What is the best way to go about designing unique primers from these data? There are many things to take into consideration, including quality of read in the area where the primers are designed, duplicate isotigs in the library, etc.
Any suggestions?
I have run BLASTX on my assembled contigs/isotigs and now have potential genes of interest that I want to design primers for doing further in-depth qPCR expression analysis on (looking at each gene over several time points post wounding).
What is the best way to go about designing unique primers from these data? There are many things to take into consideration, including quality of read in the area where the primers are designed, duplicate isotigs in the library, etc.
Any suggestions?