I need to design primers for cDNA however, the genes that i want to amplify, I am not sure if they are expressed ( i am doing a time course and they may not be expressed at the beginning but should be towards the end). This makes it hard to determine my qPCR efficiency of my primers when doing a standard curve to chose which primer pair is best to move forward with if my gene is not expressed. If i chose the best primers for genomic DNA will they still be the best primer pair for cDNA? Any thoughts? Thanks!!
Dear Kyla, the amplicon size is important parameter. If you primers amplify 90-120 bp in genomic DNA you can use. (in microbial DNA). The primers used for long amplicons are not suitable for qPCR. longer amplicons give less efficient qPCR results because more SYBRGreen is incorporated into DNA.
Best regards.
You will need to check the splicing pattern of your gene to see if your genomic DNA primers will amplify a product from your cDNA. If your gDNA primers are in introns, then you can't use them for qPCR.
Clone your gene-of-interest into a plasmid and you'll have plenty of DNA to test out your primers and make your standard curve.
The difference in genomic DNA and cDNA is that genomic DNA contains both introns and exons while cDNA only contains the exons, so as long as your primers do not include the intron region sequence, then it should be fine
The mode of designing primers is different, as qPCR primers skip introns. So, you cannot use the same primers as you use for genomic DNA amplification. The size of primers also varies. It should be of short length else the efficiency decreases.
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