I want to ask what are the benefit of treating RNA sample with DNase I before cDNA and RT-PCR? how dose that affect result !
To get rid of DNA contamination from RNA samples. If DNA is contaminated, you cannot tell whether amplified DNA is derived from complimentary DNA or contaminated DNA.
what if you design primer that are located in two exon ?! what the benefit then ?!
Hi, check this link, you may find my answer here given probably a month back. Hope it will do some help.
https://www.researchgate.net/post/How_to_clean_RNA_from_DNA_contamination
But the known fact is that any method of gDNA removal from the RNA won't guarantee at all that there will be no loss of your RNA quantity and quality, so I personally do not recommend to use DNAse treatments on extracted RNA. DNAse I is somewhat a non specific endonuclease which can introduce breaks even in DNA / RNA hybrids. So proper primer designing for qPCR has more vital role to avoid gDNA amplification. But this gDNA contamination in your samples will definitely compete for primers and / or probes later in your qPCR leading to false positive amplifications. And you can view the gDNA peaks in your melt curve if you are using the Sybr Green assay for qPCR. But if you want to use the DNAse I anyway so the best choice is DNAse I with MnCl2 in it, as this cleaves both DNA strands at same site so a better choice to use and its easily available.
- Badges
- Science topic