Hello everyone,
I am confused about qPCR primer designing using NCBI primer-blast. I designed several pairs of primers and blast results said my products were localized to mRNA of my target genes. But my collegue's results said his products were localized to genomic DNA of his target genes. As what I know, cDNA derived from reverse transcription of mRNA of target genes, so I want to know whether my primers are available for PCR or not, if not, how about my collegue's? Waiting for your answer.
Thank you so much.
- if primers are designed internally within exon they will score hits in both genomic DNA and mRNA
- if you design primers across exon junctions they will specifically target cDNA and should not return hits to clones describing genomic DNA by BLAST
As Laurenece said, the location where you designed the primers is critical. If it is within exon junctions, Primer blast will show the product on cDNA. But more importantly, when you are using NCBI primer-blast tool, you can adjust it to either mRNA or Genome, according to the figure I attached. If you choose mRNA Seq, it will blast it only with cDNA, and if you choose genome, DNA will be used for comparison.
Hope it helps you.
Your results depend on the research your are performing. If you chose mRNA database the results are RNA and if genomic DNA the results are genomic. The perfect match of your primer will be with mRNA no matter how you chose it but it will be in two pieces with genomic DNA if they are across exons junctions.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology