====Preserving field-collected samples to maintain DNA integrity====

===Introduction===
Genetic diversity experiments have become routine in many fields of biodiversity science. Despite the essential prerequisite of obtaining good-quality DNA from samples, there are surprisingly very few reviews on effective field sampling for preserving DNA before it extraction is carried out in the laboratory. Here, general recommendations are presented according to a recent review by Nagy 2010((Nagy, Z.T., 2010. A hands-on overview of tissue preservation methods for molecular genetic analyses. Organisms Diversity & Evolution 10, 91–105[[http://www.springerlink.com/content/c81h415005u27534/fulltext.html|10.1007/s13127-010-0012-4]])). The QCBS researchers that can be contacted for additional information and to answer questions are listed. Do not hesitate to contribute to this page, or to notify us of any errors or omissions by contacting [[mailto:annie.archambault@mail.mcgill.ca|Annie Archambault]]. 

The DNA molecule is stable, but sensitive to DNA-degrading enzymes (nuclease) present in the organism’s cells and in the environment. A water-free environment (highly desiccated, pure ethanol) is often sufficient to at least partially inhibit this enzymatic activity. Alternatively, a deep freezing temperature, or a liquid buffer solution can also denature and inactivate nucleases, thereby preserving DNA integrity until the time of extraction. Methods for collecting biological samples are highly dependent on the organisms, the type of tissues (solid, soft, fluid), and on fieldwork constraints (reviewed in Nagy 2010((Nagy, Z.T., 2010. A hands-on overview of tissue preservation methods for molecular genetic analyses. Organisms Diversity & Evolution 10, 91–105[[http://www.springerlink.com/content/c81h415005u27534/fulltext.html|10.1007/s13127-010-0012-4]])) ). For long-term storage, temperature is the main factor influencing DNA preservation before it is extracted from the organism tissues.   

===General recommendations for sample collection=== 
During fieldwork, two different collections should be prepared for each sample: one for museum collections, and another for DNA extraction. As mentioned in Nagy 2010((Nagy, Z.T., 2010. A hands-on overview of tissue preservation methods for molecular genetic analyses. Organisms Diversity & Evolution 10, 91–105[[http://www.springerlink.com/content/c81h415005u27534/fulltext.html|10.1007/s13127-010-0012-4]])) ), critical questions have to be addressed before fieldwork. 
  *Is cooling/freezing/buffering/drying possible during the field expedition? 
  *What are the options and costs for transportation, and are they coupled with special permit procedures and/or safety risks? 
  *Is it possible to prepare a working stock of tissues and a backup stock (for cases of emergency)? 
  *Is it planned to isolate DNA from fresh material immediately after collection?

<WRAP round important>The best collection practices and preservation methods should be evaluated and tested in the laboratory **prior to field work**, to confirm that good quality DNA can be efficiently extracted from the collected samples. All samples, except those dried with silica gel should be placed in cold or freezing temperate as soon as possible.</WRAP> 

===Guidelines for preserving DNA integrity in biological samples===  
The guidelines listed below, organized according to the type of organism, follow recommendations from Nagy 2010((Nagy, Z.T., 2010. A hands-on overview of tissue preservation methods for molecular genetic analyses. Organisms Diversity & Evolution 10, 91–105[[http://www.springerlink.com/content/c81h415005u27534/fulltext.html|10.1007/s13127-010-0012-4]])) and from QCBS members and students. In addition to the diverse methods developed, universal commercial solutions, such as [[http://www.qiagen.com/products/rnastabilizationpurification/rnalaterrnaprotectsystems/rnalaterrnastabilization.aspx#Tabs=t0|RNAlater]], are now available for any type of samples or organisms. The RNAlater solution is reported to be made of 25 mM Sodium citrate trisodium salt dihydrate, 10 mM EDTA, 70 g/100 ml Ammonium Sulfate; adjusted to a pH of 5.2 with 1M H<sub>2</sub>SO<sub>4</sub>.   

==Small to large animals== 
A small tissue sample can be removed from any part of the body that is not critical for identification or research purposes. Blood samples, buccal swabs, finger clippings (frogs), tail clippings (rodents or fishes) can be sources of good quality DNA. Non-body parts, such as feathers, hair, faeces, foot (snails) or body mucus (fish) can also be used as sources of DNA. The most commonly used DNA preservation buffer for animal samples is ethanol, to which substances inhibiting enzymatic activity (e.g. EDTA or 1–3% glycerine), and chaotropic agents may be added. The optimal ethanol concentration is about 95–99%. Traces of benzene in 100% ethanol can affect DNA preservation, while 70 % will lead to DNA degradation.  These preparations are, therefore, not recommended. The ethanol-tissue ratio should exceed 5:1. In order to maintain the ethanol concentration to at least 95%, it is necessary to replace and control the ethanol solution within the first days (at least three days) after sampling, and vial should be sealed perfectly with Parafilm. Ethanol should be removed prior to DNA extraction. 

Different methods to preserve animal samples also exist. Tissues can be preserved in various storage buffers (CTAB; TNES((TNES: 2M Tris-HCl, 0.5M EDTA, 5M NaCl and 20% SDS at pH=8.0)); TNE2((TNE2: 10 mM Tris-hydroxymethyl-amino-methane, 10 mM NaCl, and 2 mM EDTA at pH=8.0)); Guanidinium isothyocyanate). Very small animal tissues or fluids (blood samples, saliva, sperm) can be desiccated using drying paper((FTA paper®, Whatman)) or silica gel (amphibian and fish tissues). 

Many QCBS researchers use DNA-based methods for studies on animal biodiversity, and the methods to they use to preserve samples are briefly listed. The choice storage buffer is 95% to 99% ethanol, and researchers stress the importance of maintaining a high ethanol:tissues ratio, and on replacing ethanol frequently during the days following sampling. Very different organisms can be preserved in ethanol solution: whole insects ([[http://qcbs.ca/members/main-researchers/?profile=10|Ehab Abouheif]], [[http://qcbs.ca/members/main-researchers/?profile=57|Jade Savage]], [[http://qcbs.ca/members/main-researchers/?profile=67|Terry Wheeler]]); small piece of adipose fins or caudal fin from fishes caught by electrofishing or commercial minnow traps ([[http://qcbs.ca/members/main-researchers/?profile=35|Dany Garant]], [[http://qcbs.ca/members/main-researchers/?profile=11|Bernard Angers]]), whole body of small snails ([[http://qcbs.ca/members/main-researchers/?profile=11|Bernard Angers]]), piece of gonad and surrounding foot epithelium from freshwater mussels  ([[http://qcbs.ca/members/main-researchers/?profile=11|Bernard Angers]]), tail tips, eggs or larvae from salamanders ([[http://qcbs.ca/members/main-researchers/?profile=11|Bernard Angers]]), tail tips or whole skinned body from small mice ([[http://qcbs.ca/members/main-researchers/?profile=45|François-Joseph Lapointe]], [[http://qcbs.ca/members/main-researchers/?profile=52|Virginie Millien]]) or small mammals ([[http://qcbs.ca/members/main-researchers/?profile=35|Dany Garant]]).

Other preservation methods,without ethanol, are also used. For instance, DNA from birds or mammals can be preserved until extraction by impregnating blood in a 25 mm2 piece of filter paper ([[http://qcbs.ca/members/main-researchers/?profile=35|Dany Garant]]). Zooplankton, on the other hand, are typically collected with nets (80 um mesh), and can be preserve with 5% sugar-formalin, as well as in 95% ethanol ([[http://qcbs.ca/members/main-researchers/?profile=30|Alison Derry]])


==Plants==
Optimal DNA quality and quantity is obtained from fresh young plant tissues. For field sampling however, a plant sample is usually a leaf dried in silica gel beads.  Rapid desiccation of the tissue inhibits the nuclease's enzymatic activity. For optimal use, the ratio of silica to sample should exceed 10:1, the tissue dissected in small pieces, and material checked for dryness regularly. Although less common, anhydrous calcium chloride (CaCl2) could also be used for desiccation. Plant samples can also be preserved in CTAB buffer (Hodkinson et al. 2007 ((Hodkinson, T.R., Waldren, S., Parnell, J.A.N., Kelleher, C.T., Salamin, K., Salamin, N., 2007. DNA banking for plant breeding, biotechnology and biodiversity evaluation. Journal of Plant Research 120, 17–29 10.1007/s10265-006-0059-7|http://www.springerlink.com/content/h6tv0816367tq45n/]])) ), or in guanidinium isothyocyanate liquid buffer, which denatures RNA, DNA and proteins. 

Many QCBS researchers are botanists ([[http://qcbs.ca/members/main-researchers/?profile=27|Selvadurai Dayanandan]], [[http://qcbs.ca/members/main-researchers/?profile=65|Marcia Waterway]], [[http://qcbs.ca/members/main-researchers/?profile=59|Daniel Schoen]], [[http://qcbs.ca/members/main-researchers/?profile=85|T. Jonathan Davies]]), and a lot are based at the [[http://www.biodiversite.umontreal.ca/|Centre sur la biodiversité]] ([[http://qcbs.ca/members/main-researchers/?profile=74|Simon Joly]], [[http://qcbs.ca/members/main-researchers/?profile=19|Anne Bruneau]], [[http://qcbs.ca/members/main-researchers/?profile=18|Luc Brouillet]]) where modern biobanking facilities exist. For most of the botanical collections, the choice method for preserving DNA integrity between the field and the lab is silica-gel drying using high quality plastic bags (e.g. Ziploc). Researchers stress the importance of harvesting the youngest leaves, of keeping a high silica:tissues ratio, and of sealing vials or bags tightly.   

==Environmental or fungal samples== 
For environmental samples, such as soil core or rhizosphere, a variety of methods exist for preserving DNA integrity between field sampling and laboratory DNA extraction. When compared, the non-toxic sucrose lysis buffer (SLB) surpassed the guanidine isothiocyanate (GIT) buffer for preserving DNA from sediment samples stored at room temperature for a week Mitchell and Takacs-Vesbach 2008 ((Mitchell, K.R., Takacs-Vesbach, C.D., 2008. A comparison of methods for total community DNA preservation and extraction from various thermal environments. Journal of Industrial Microbiology & Biotechnology 35, [[http://www.springerlink.com/content/w638035028100452/|1139–1147. 10.1007/s10295-008-0393-y]])). Other sample storage buffers available are a CTAB buffer in saturated NaCl, 200 mM sodium ascorbate on samples previously homogenized in a sorbitol wash buffer ; or a TNES buffer (Nagy 2010((Nagy, Z.T., 2010. A hands-on overview of tissue preservation methods for molecular genetic analyses. Organisms Diversity & Evolution 10, 91–105[[http://www.springerlink.com/content/c81h415005u27534/fulltext.html|10.1007/s13127-010-0012-4]])) ). 

Many QCBS researchers routinely analyze genetic diversity of fungi and of microorganisms from environmental samples. When based in the arctic, permafrost core are simply maintained frozen until DNA extraction can take place ([[http://qcbs.ca/members/main-researchers/?profile=68|Lyle Whyte]]). In warmer areas, soil samples are commonly sieved on site, kept in plastic bag on ice packs in a cooler for the day, then frozen ([[http://qcbs.ca/members/main-researchers/?profile=72|Yolande Dalpé]], [[http://qcbs.ca/members/main-researchers/?profile=42|Mohamed Hijri]], [[http://qcbs.ca/members/main-researchers/?profile=60|Marc St-Arnaud]]). Samples can also be instantly frozen in ethanol poured over on dry ice, in a cooler, or preserved in a commercial  [[http://www.mobio.com/soil-rna-isolation/lifeguard-soil-preservation-solution--100ml.html|LifeGuard™ Soil Preservation Solution]] that will enable separation of soil from rhizosphere.