How biological materials (Animal carcasses, excrements) collected in field can be best stored for a later DNA and RNA extraction? what is the protocol to follwo and what are the storing equipements needed.?
Hi Imad,
We use a "homemade" buffer in our expeditions, where freezing at -80 is not an option. It is called NAP buffer and preserves well DNA and ARN. Here is a link to the article: https://onlinelibrary.wiley.com/doi/pdf/10.1111/1755-0998.12108?casa_token=qgIKvJ4dV1kAAAAA:sEJG807qo-hjPXIc9J54AKwwkxiiqsZSWmFdTd91VobTRfivCVxUYECwRzyUByrGGabSVsPZHrdl. We usually collect tissue samples of liver from carcasses, when it is not fresh, muscle/heart works better, given that liver degrades quick. For mammal specimens that we collect, we skin them in situ and fix the skin in formalin for a few hours and then store it in ethanol 70%, but the skinned body goes directly to ethanol 70% so that there is still a good stock of DNA. For feces Carlos Dominguez Sarabia might know better. Once we get things back to our institution, we store the samples in a -80 ºC freezer.
Cheers,
Arlo
It depends on what type of biological materials you want to store. If you're going to store regurgitated pellets of raptor birds or scats or droppings of mammals, you can use the Sundry method ( 35 Celcius to 40 Celcius) or air dry method. You can prevent moisture by putting them into plastic Ziploc bags with silica gel.
If your goal is to store some tissue samples, then I must say, use standard protocol according to your needs and target organisms.
1) For insects collected for DNA extraction are stored in ethanol.
2) For the preservation of Insect Haemolymph, It should be collected in ice-cold Eppendorf tubes with a sterile ice-cold anticoagulant buffer.
3)For bird feathers, samples can be stored in clean Ziploc bags and freeze them. avoid the use of ethanol or any other chemical
4) If you have tissue samples for DNA/RNA extraction, it is advisable to keep it in pure or 90% alcohol.
Among the most often used preservation method of samples collected for DNA analyses is freezing. Freezing at −80 °C or in liquid nitrogen (−196 °C) is most often used for long term storage
https://www.zymoresearch.com/blogs/blog/tips-tricks-for-rna-isolation
https://pubs.usgs.gov/tm/15/c04/pdf/tm15-c4.pdf
Cryo-preservation is best method for sample tissue carcasses and excrements preservation for long duration.
Hi Imad,
We use a "homemade" buffer in our expeditions, where freezing at -80 is not an option. It is called NAP buffer and preserves well DNA and ARN. Here is a link to the article: https://onlinelibrary.wiley.com/doi/pdf/10.1111/1755-0998.12108?casa_token=qgIKvJ4dV1kAAAAA:sEJG807qo-hjPXIc9J54AKwwkxiiqsZSWmFdTd91VobTRfivCVxUYECwRzyUByrGGabSVsPZHrdl. We usually collect tissue samples of liver from carcasses, when it is not fresh, muscle/heart works better, given that liver degrades quick. For mammal specimens that we collect, we skin them in situ and fix the skin in formalin for a few hours and then store it in ethanol 70%, but the skinned body goes directly to ethanol 70% so that there is still a good stock of DNA. For feces Carlos Dominguez Sarabia might know better. Once we get things back to our institution, we store the samples in a -80 ºC freezer.
Cheers,
Arlo
Pour l'ADN il n'y à pas de souci vous conservez vos échantillons directement dans le formol ,mais l'extraction de l'ARN vaut mieux congeler l'echantillon
The best thing you can do is to use RNAlater for RNA in the field sampling. In the laboratory preserve it at -80 and to manipulate it before carrying out the extraction use liquid hydrogen
The use of some freezing technique like liquid nytrogen is not practical in field conditions. It would be better to use some stabilization agent, for example
Qiagen Allprotect Tissue Reagent or RNAlater. These allow to keep the samples to room temperature for some days until freeze them, but have the disavantage of the price. Is more cheap to use some "homemade" buffer, as recommended other colleagues.
Hi Imad,
For feces, a solution of 80% is optimal for DNA preservation (check out http://abc.museucienciesjournals.cat/volum-43-2-2020-en/towards-high-throughput-analyses-of-fecal-samples-from-wildlife/?lang=en). If your scats come from arid or semi-arid landscapes (for example, the Sahara desert), a paper bag might be enough. And as Arlo Hinckley pointed out, there are home-made buffer solutions for other kinds of material.
Greetings!
Dear Sidi Imad Cherkaoui,
Blood is the best sample to collect for DNA extraction from dead birds (if the bird is freshly deceased or has been frozen soon after death). If no blood is available, brain or liver are the best tissues to collect from bird corpses. The tissue should be diced and stored in ethanol (i.e. Analytical Reagent(AR) grade ethanol, not more than 1 to 10 ratio of tissue to ethanol and stored in rubber sealed tubes). Sterile cotton swabs should be used to collect samples of feaces, pus, heart blood and other body fluids. These swabs should be transported under refrigerator or in the appropriate medium. The gastrointestinal tract are best handled by obtaining a loop tied at both ends and placed in a sterile polythene covers. Monitoring of genetic variation by modern techniques such as DNA fingerprinting is very important in this regard. Non-invasive methods of DNA extraction and the universal primer for species identification which is widely popular owing to its successful application in solving wildlife forensic cases since more than a decade has also been developed. The techniques included in this category are storage of DNA, embryos, or captive breeding through the establishment of Gene Banks.
Ashish
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