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--- sample_collection_and_dna_extraction [2012/02/22 11:49]
anniearchambault
+++ sample_collection_and_dna_extraction [2012/02/22 14:28]
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 ==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 sourcesof DNA. The most commonly used DNA preservation buffer for animal samples is ethanol, where 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, and these 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.
+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, where 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, and these 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 dessicated 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 accepted to share their sample preservation methods. 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. This is how tissues from different organisms can be preserved: 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]]).
+Many QCBS researchers use DNA-based methods for studies on animal biodiversity, and the method to they use to preserve sample is 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 than 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]]).
+Other preservation methods than 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]])
-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==
-Most commonly, plant sample is a leaf that is dried in silica gel beads, which, by rapidly desiccating the tissue, inhibit the nucleases enzymes activity.
+Optimal DNA quality and quantity is obtained from fresh young plant tissues. For field sampling however, a plant sample is most commonly a leaf that is dried in silica gel beads, which, by rapidly desiccating the tissue, inhibit the nucleases enzymes 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 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 denaturates RNA, DNA and proteins.
-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 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 denaturates 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. Optimal DNA quality and quantity is obtained from fresh young plant tissues. For most of the botanical collections however, 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.
+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]])).
+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 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]]).
+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.