Difference between revisions of "Tissue Sample Collection"

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	# Tissue prepared this way and kept in LN<sub>2</sub> can be stored indefinitely and later transported to a lab with experience in tissue culture to establish cell lines.		# Tissue prepared this way and kept in LN<sub>2</sub> can be stored indefinitely and later transported to a lab with experience in tissue culture to establish cell lines.
	[[File:Slide29.jpeg|center|Comparison of most commonly used preservation methods by Zimkus (unpublished).]]		[[File:Slide29.jpeg|center|Comparison of most commonly used preservation methods by Zimkus (unpublished).]]
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−	== Discipline Specific Information ==
−
−	=== Herpetology ===
−	* Liver and skeletal muscle are perhaps the most commonly sampled tissues.
−	::- A small incision can allow researchers to push the liver out, causing minimal damage to specimens being used for morphological study.
−	::- Bile salt can contaminate this organ and affect tissue stability, so tissue should be preserved as soon as possible and the gallbladder avoided (Dessauer et al. 1990).
−	* Muscle can be dissected from the thigh on one side, leaving the remaining side intact for morphology, but it has been reported that yields are small due to tough fibers (Gamble 2014; Wong et al. 2012).
−	* Non-lethal sampling methods may include (Ezaz et al. 2009; Gamble 2014; Mollard 2018; Mollard et al. 2018):
−	::- Biopsies
−	::- Blood draws
−	::- Feces collection
−	::- Skin swabs (often used to test for chytrid fungus infection)
−	::- Sperm or spawn collection (hormonally-induced)
−	::- Toe clips
−	::- Tail clips
−
−	=== Ichthyology ===
−	* Take tissue clips from right side (as for all destructive samples)
−	* Fin
−	* Muscle (hypaxial)
−	* Gill
−	* Whole animal (e.g. larval fish)
−	* Eye (especially from larvae), from right side
−
−	===Mammalogy===
−	* Camacho-Sanchez (2013) found that rat (''Rattus rattus'') liver yielded the best RNA and DNA quality when compared to blood, brain, ear clips, muscle, and tail tips.
−
−	==References==
−	<references/>
−
−	[[Category:Desired BP Content]][[Category: Field Collecting]][[Category: Preparation]]

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Revision as of 18:48, 13 May 2019

Statement of Purpose

These links and documents contain information about best practices for tissue sample collection.

Introduction

Numerous methods are currently being used to preserve tissue and likely depend on the specific aim of a scientific study or goals of an institutional or collaborative program (e.g., biobank, multi-institution initiative). Samples may be collected for individual research projects with explicit and relatively short-term goals (e.g., molecular ecology, molecular phylogeny, population genetics). Studies may also be taxonomically or regionally focused, such as rapid biodiversity assessments that use DNA barcoding techniques to identify species surveyed in a specific region. In contrast, biobanking initiatives or collaborative programs involving multiple institutions may have targeted specific species for long-term conservation and/or use in Assisted Reproductive Technologies (ARTs). The ultimate aim of a research study or conservation initiative may dictate the specific tissue types or biomolecules needed to fulfill the project goals. Molecular studies traditionally used DNA as it could be preserved more easily in the field with many methods. Unfortunately, the various methods used for DNA preservation are not equally effective, and DNA may be fragmented or otherwise compromised. Some preparations may allow high-quality Sanger sequencing reads but prevent high-quality gDNA needed to sequence genomes or the high-molecular-weight DNA needed for long-read sequencing and other technologies (e.g., BAC library preparation, optical mapping, 10X Chromium libraries). RNA is increasingly being used in gene expression studies but is preserved using fewer methods and degrades rapidly if not frozen immediately. Researchers should, therefore, consider all preservation options as some may allow them to both fulfill their study goals and aid in current or future research or conservation initiatives. In addition, those Researchers collecting samples need to consider the location of initial preservation and if possible carry out a feasibility study to ensure that the selected preservation method(s) will work given any logistical constraints.

Contributors

Major editor: Breda Zimkus. Original content generated during The American Society of Ichthyologists and Herpetologists (ASIH) Annual Joint Meeting - 2016, during an iDigBio sponsored workshop by the following individuals participating in the "Field to Database" Group of the aforementioned workshop: Breda Zimkus, Cesar Aguilar, Ben Frable, Meredith Mahoney, Zachary Randall, and David Wernecke.

Vial Considerations

One area of immense variability is with the number of vial types used in natural history collections ^[1].

• Samples stored using LN₂ must have vials that are rated for use with cryogenic temperatures, preferably made of polypropylene with screw-top caps. Glass vials are problematic when frozen because of their fragility when handling and vulnerability to cracking when stressed, and their use with LN₂ is unacceptable because leakage into the vials can lead to the vessels exploding.
• Pop-off lids are not recommended because this vial type can easily open on its own.
• Vials can have threads located internally or externally on the vial opening; both vial types exhibit advantages and disadvantages.

- Externally-threaded closures promoteb more sterile conditions because internal threading can allow contaminants to enter if the cap is placed on an unclean surface when removed, but these vials can be susceptible to cracks and loss of air-tightness, which can lead to sample dessication or oxidation (Corthals and DeSalle 2005, Corthals 2006).

-Internally-threaded vials might allow increased storage capacity, depending on the vial selected, but users also suggest that material can be trapped within the threads of this vial type (Johnson 1999).

• Some manufacturers suggest that vial caps that incorporate a silicone gasket or O-ring (internally or externally threaded) are ideal for vapor-phase LN₂ freezing because the seal is enhanced; however, there are some concerns:

- Care must be taken if the vial cap is over-tightened because the gasket can become distended.

- The presence of gaskets or O-ring might improve the initial performance of seals but can be problematic when used with some alcohols (e.g., ethanol) because some gasket material, such as silicone, is vapor permeable.

• When working with liquid-phase nitrogen cold storage, extra caution must be taken because the accidental entrapment of liquefied nitrogen inside the vial leads to pressure build up and, upon removal, rapid vaporization of the liquid can result in leakage or even explosion.

- As a precaution, vial manufacturers recommend that samples not be immersed in LN₂ unless they have secondary containment. Heat-sealing vials into flexible polyethylene tubing is recommended for safe storage in the liquid-phase environment.

Labeling and organizing vials

• Pre-label vials

-Many researchers include specimen tags inside vials, but paper inside vials not ideal because of possible contamination

• Use alcohol-proof pen
• Use scribe to etch identifying information as back-up
• Organizing vials numerically in boxes is preferable to storage in bags in case writing comes off vials
• Tissue samples should be taken as soon after euthanization as possible

Tissue Collection Methods

Some general recommendations include:

• Ensure that gloves are worn
• Make sure that all instruments and surfaces are sterilized (or use disposable products) between sub-samples
• Collect sample as soon after euthanasia as possible

-Pre-labeling vials may increase preparation speed

• Fill two sets of tissue vials in the field: one for current project and one for institutional collection (rather than subsampling one vial later).
• Label vial to indicate unique number and tissue type

- Only place one tissue type in each vial

- Epigenetic studies require same tissue type for all samples in study (i.e., all muscle)

• Cut/slice up tissue sample, small size allows permeation by preservative (Note: read specific recommendations for preservative provided by manufacturer.)
• Fresh preservative may be needed if in field for extended period of time
• Don’t over-fill vial with tissue, or tissue and preservative (expansion, can interfere with vial threads, etc.)

Tissue Preservation Methods

The methods currently used to preserve genetic material during initial sampling vary widely owing to the sampling location, tissue type, and intended research use (Dessauer and Hafner 1984, Prendini et al. 2002, Bus´ and Allen 2014). A comprehensive treatise of methods to preserve genetic samples is discussed in Nagy (2010). In a survey of genetic resource collections associated with natural history

museums, samples were found to be initially preserved in a variety of different ways ^[1]

1. ↑ ^{1.0 1.1} Zimkus, B.M. and L.S. Ford. 2014. Genetic resource collections associated with natural history museums: A survey and analysis to establish a benchmark of standards. Pp. 9–44 in DNA Banking for the 21st Century. Proceedings of the U.S. Workshop on DNA Banking (W.L. Applequist and L. Campbell, eds.). William L. Brown Center, St. Louis, Missouri. 187 pp.