I don't know, in my study model (human cell line), which transcript variant is going to be expressed.
Hi Deepa, then the only answer can be: Design primers from the variants and check which one will amplify. All the best, Nadine
I agree, you need to construct several primer pairs. In addition, I would make multiple sequence alignment with all variants and determine highly conservative regions. Then pick up primers from these regions. If transcripts are with SNPs you will get one size products in qPCR from all possible variants.If you sequences contain indels, you will see size variations in melt curve analysis.
Hi, you can design primers in the exon exon junction common to all transcript variants.
When there are many variants of a gene it is better to use a consensus sequence with degeneracy while designing the primers so that it will effectively capture all the variants
Design your primer after multiple sequence alignment of cDNA of all transcripts and go for e-PCR for initial prediction. I think this will help u to design the best primer for the particular sequence.
Hi Deepa, in other way, you can try PrimerDesign software in web Universal Probe Library by Roche Lifescience,
After you have several option of transcript varian, u can choose common assay at the bottom to make the software generate primer and probe that will have the same amplicon sequence, so u can have 1 primer probe to all transcript varian.
Cheers
Hi,
If you are not interested in investigating which variants are highly expressed or most conserved regions and nucleotide variation in your cell line. You have to choose any one of the largest size (mRNA sequence) of the variant for qRT-PCR primer designing. It will amplify good ...
Can you please guide me, i want to design a primer for lets say gene A, this gene has not been studied in species of my interest, How i can design primer in such conditions??
Hi Qaisar Shahzad,
You need to know the sequence of the gene that you want to study in order to design the primers. You can send the gene for sequence. However it´s also necessary for you to know the nucleotide sequence of the organism in cause to check if the primer is specific for that intented target or if it amplifies any other target
- Badges
- Science topic