Isolation of both DNA and RNA from the same plant tissue - (Mar/26/2012 )

Dear colleagues,

I would like to ask, whether you have experiences with isolating both DNA and RNA from the same tissue. I am working with plants. I tried some commercial kits - basicly, the RNA isolation kit is used with extra buffers for extracting DNA. The RNA extraction part works perfectly, but I have very low amounts of DNA. Thus, it makes no sense to use this approach for isolation of both nucleic acids. I was then thinking about isolating DNA and RNA separately, with the specific kit or non-kit method. The problem is, how to split my tissue, since I would like to have nucleic acids from the same tissue. My idea was to:
1.) homogenize the sample - I am always using leaves frozen in nitrogen, and homogenizing with metal beads in a homogenizer.
2.) add a lysis buffer to this frozen powder, make the sample liquid, and then split into two parts, and continue with the extracting DNA and RNA separately.

However, the question is, whether I can use the lysis buffer from e.g. RNA kit, and then to continue with isolating DNA by DNA extraction kit, or eventually vice versa, i.e. using lysis buffer from DNA extr. kit, and continue with isolating RNA by RNA extr. kit. I contacted the manufacturer of the kit I was using (Macherey Nagel), and they said that this is not possible, since the lysis buffers for DNA and RNA extraction are different. I tried anyway, and it somehow works, but I would like to have a kind of representative results
I am wondering whether there is some better way how to split my samples into two parts, so I can then proceed to DNA and RNA extraction without affecting it negatively? Thanks a lot in advance

Did you try Trizol? (or TriReagent and several other names) It's a phenol based reagent for simultaneous isolation of RNA, DNA and proteins (but you don't need to do all) from different fractions. It also lyses and homogenises the sample.

Hi Trof, thanks a lot for reply. Yes, I tried Trizol - for RNA works perfectly, for DNA not... I experienced difficulties with extracting good quality DNA - I got degraded DNA Moreover, with Trizol, you are extracting the DNA from the mess (i.e. the cell debris) that is left after taking the upper phase containing RNA, and you are working with this debris until the very end, so I cant help myself - to me it seems so unpure, that I am wondering whether I can get a pure DNA from that. At the very end of the protocol, DNA is resuspended with 8mM NaOH, and if one wants to get rid of the cell debris, it is possible to centrifuge it and pipet the supernatant. Do you have experience with extracting the DNA using the Trizol? I want to use the DNA for qRT-PCR.
Anyway, I will give the Trizol one more chance, but I would prefer some more pure method to get both DNA and RNA

I did the isolation using Trizol, and for the DNA isolation I found a modified protocol using Back Extraction Buffer based on guanidium thyocyanate. It worked very well. If anybody is interested, I can provide the protocol that I found somewhere else.

talianka on Wed Apr 4 08:40:24 2012 said:

Its a protocol using the DNA and RNA from the same sample then or?

pito on Wed Apr 4 21:26:13 2012 said:

Yes, it's using the organic phase left after pipett out the RNA, but DNA is extracted by different method. This alternative protocol is even part of the Invitrogen product note, though I didn't know about it until now.

pito on Wed Apr 4 21:26:13 2012 said:

Yes, exactly as Trof explained. But for the extraction of DNA from the organic phase, I finally did not use the protocol described by the manufacturer, because I had very bad experience with the quality and purity of the DNA. Instead, I found another protocol enabling to extract the DNA of good quality from the organic phase left after RNA extraction.

Different even than the Back extraction protocol in the Alternative procedure for DNA isolation part of the document I linked? Are there other back extraction protocols that differ from this one?

Sorry that I am answering so late - it is the same protocol,as you linked, Trof