I have old samples that may have been fixed in formalin, or not, there is no way to know, and then preserved in alcohol. I'm afraid the DNA extraction protocol for samples fixed in formalin may be overly negative if the samples have been originally fixed in alcohol. What is the least risky path to follow? Any suggestion for protocols that have a general use?
Thank you!
i had samples that were conserved in all protect from qiagen for 6 months and i didnt get lucky enought to extract any DNA so if possible i always recommand to work with fresh tissue but i know that machrey nagel propose spécial kit for tissues and i got the best quantity and quality when using them ( i already tried promega and trizol and dnazol and qiashreeder special tissue of qiangen) so good luck!
hi
Article High quality DNA obtained with an automated DNA extraction m...
In my area of research, we commonly extract pathogen DNA that is embedded in animal tissue. DNA can be detected in museum specimens from the 1800's that were fixed in formalin and preserved in ETOH. I use the Qiagen DNeasy kit, but other kits may work just as well, such as ThermoFisher Purelink Genomic DNA. It is imperative that all of the ethanol is evaporated off the samples before you begin the extraction process. DNA can then be detected using a real-time Taqman PCR.
Hi Marina, Have you extracted/detected DNA from museum specimens from the 1800's that were fixed in formalin and preserved in ETOH?
You have stated that you use Qiagen DNeasy kit, and suggesting other kits such as ThermoFisher Purelink Genomic DNA! Have you used these kits for old samples that may have been fixed in formalin, that was the question asked here, Thanks!
Depends on the temperature it is stored as well as the sample type of which you want DNA . I would suggest you to use Thermo fisher purelink Genomic DNA Kit.
Thank you very much for all the suggestions! It's frog tissues, and they were not preserved at -80 sadly.
Thank you Zeena, that's an interesting paper, it gives a lot of ideas.
Marina, which quantity of sample would you recommend using as a minimum? I know the draw back between sample size and final concentration, but I'm afraid Quiagen may not work, and then I would like to have enough tissues for a second round. The problem is, I I don't have too much tissue available.
Thank you!
Hi Amaël Borzée,
Whole specimens are not normally stored in a -80, so that's okay. There is a standard protocol for quantifying Bd DNA by swabbing amphibian specimens (5x on ventral surface, 5x between toes, etc.), and I have always used this method. If you have less skin sample than that, I would not be sure how to proceed.
Detecting Bd in formalin fixed amphibian tissue is a common practice though. The quantity is likely lower than fresh tissue, but nonetheless, it is detectable. Basically, formalin does degrade DNA into short strands, but fortunately, qPCR (especially using Qiagen DNeasy blood and tissue kit for extraction rather than Prepman Ultra) is extremely sensitive, and can use short strands to amplify. It probably has to do with the fact that we are using a very short but specific sequence of DNA from Bd and that the gene region we use is also prolific in Bd DNA (so there are many copies). Most of the problems with retrieving DNA from formalin fixed tissue is when you need long strands of DNA (such as with ITS sometimes). The formalin causes the DNA to break and form primer dimers, basically it gets all jumbled up and stuck together: difficult for studies that require long strands of DNA. It is amazing that Bd DNA survives so well in formalin fixed animals.' Most of the time, specimens that were fixed in formalin were only exposed for a short time, then transferred to ethanol for the long haul, so there is a good chance that some of these gene regions are still in tact in at least some of the cells. It turns out that regular PCR is not really sensitive enough, but after cleanup (DNA digestion and extraction), qPCR can find and amplify such short fragments (
Thank you very much Marina! I'm actually looking at extracting the genomic DNA of the individual, it's tissues so not working on Bd for that project. But thank you for all the explanations, it should work the same for frog mtDNA.
Amael
Have you succeded in DNA extraction? I'm looking for a DNA extraction protocol from Invertebrate dry tissue, formerly fixed in formalin.
Cynthia M. Asorey it worked in the way that I got DNA, but sequencing results are not clean. It was for species identification, so it worked out after some guesses and deduction, but I wouldn't use my sequences for any analysis. I can send you the protocol I use if you want.
Amaël Borzée yes, pls! I will appreciate if you send me your protocol. There is a chapter about it in the Ancient DNA book (Springer, 2012). These authors suggest a pretreatment with alkali and heat, but the whole protocol doesn't include extraction with a kit.
- Badges
- Science method