I'll be surprise that you get pure RNA without DNA contamination as most of the dual extraction kits which separates both nucleic species always give some genomic DNA in the purified RNA and you need sometimes to do two DNAse treatment to really avoid any unwanted reaction of the DNA specially in Q PCR.

Most of the kits use classical silica membrane which binds all nucleic acid.

Looking more deeply I found an intersting comment from Karen A. Darbinyan on another topic and here is what she comments #KarenADarbinyan

" Hello, Spin column-based nucleic acid purification is a solid phase extraction method to quickly purify nucleic acids. This method relies on the fact that nucleic acid will bind to the solid phase of silica under certain conditions. RNA is structurally very different from DNA. RNA is single-stranded, while DNA is mostly double-stranded. If the pH and salt concentration of the binding solution are optimal, the RNA will bind to the silica gel membrane as the DNA passes through. Than you can elute RNA from column Hope this info will help you. Good luck! "

Thank Jean-Pierre Dangy for your answer. That's exactly what i don't understand. The kit claims it includes RNase-free lysis and wash solutions that protect RNA from RNases while liberating the RNA from DNA, proteins, and other cellular debris without the need for further DNase treatment (very low minimal residual DNA carry over) so i have no idea how do i get pure RNA without DNase treat. and i did NanoDrop, my purification result shows 260/280 ratio = 2.02 does that mean 'pure RNA?'

(It's my first time working with lab technicians with no prior experience in Q PCR since i have only learned about clinical studies so it's quite difficult to understand for me )

Dear Sukpurn that looks good to me here is a Nanodrop insight on nucleid acid purity

" 260/280

The ratio of absorbance at 260 nm and 280 nm is used to assess the purity of DNA and RNA. A ratio of ~1.8 is generally accepted as “pure” for DNA; a ratio of ~2.0 is generally accepted as “pure” for RNA. If the ratio is appreciably lower in either case, it may indi-cate the presence of protein, phenol or other contaminants that absorb strongly at or near 280 nm "

Oh my bad! i didn't notice the 'read more' and i skipped your previous answer from #KarenADarbinyan This really helps me out. Thank you so much Jean-Pierre Dangy for your time i'm really grateful for your help.

You're most welcome

RNA is a single strand, while DNA is a double strand molecule. At the optimum pH and salt concentration of the binding phase, RNA adheres to the silica gel column membrane, where it could be recovered and the DNA filters through.

Dear all.

One last thing about this discussion is to remind you that DNA molecules are bigger than the RNA ones. Your qPCR primers may be designed to cover exon-exon junctions and, in this case and because your amplicons will be very short, DNA will not disturb your reaction and goals during the qPCR.

So, even If your extraction was not good enough, it would probably work just as fine as it was free of DNA.

All the best.

Hi,

As I understood, DNase is not included in the kit. So there is a possibility that DNase is added into the wash buffer content or the filter whole size is proper to purify the RNA. C. Sukpurm Could you please put your gel picture here to verify your results?

thank you for your answer Maryam Sargolzaei that's very kind of you. I did not run the PCR product on a gel i will be using RNA for cDNA synthesis for qPCR.