qPCR, Appearance of amplification curve for my minus RT control
Posted 15 September 2012 - 04:27 PM
Thanks in advance
Posted 15 September 2012 - 07:11 PM
Posted 16 September 2012 - 08:39 AM
Posted 16 September 2012 - 10:27 AM
Typically this is dealt with by treating extracted RNA with DNAse enzyme. If you gene has introns, then using primers that span the intron can also be an effective technique to differentiate cDNA (from your RNA sample) from genomic DNA contamination.
Posted 21 September 2012 - 11:41 AM
mRNA has the same sequence as DNA but misses the introns that were cut off. You designed primers for mRNA (that's complementary to cDNA), if you have both primers in the red exon for example, the primers will amplify cDNA but also the DNA if it's present and you can't tell the difference.
By designing one primer in the green exon and the other one in red, only cDNA will give you desired length, the product from DNA would be longer so you can distinguish them. If the intron between the two exons is very long (i.e. longer than say 1000-2000 bp) the DNA would not amplify at all. This is called "span the intron".
Also by designing one of the primers on the exon-exon boundary (like half of the primer in the green exon, second half in red exon) would cause that the primer won't even bind to DNA, so you will have no amplification from DNA.
These two techniques are used to prevent amplification of trace DNA in your cDNA samples. Also it's better to treat the mRNA with DNase, but that sometimes isn't 100% sure. RT- control is for checking you don't have DNA in your cDNA sample.
I never trust anything that can't be doubted.