Robert Hanner, Ph.D.

Coriell Institute for Medical Research

Last updated: 31 January 2005

1. Introduction

The purpose of this commentary is to give readers a brief update on modern principles and challenges of collection, storage and use of genetic resource collections (GRCs) as the relate to ongoing projects associated with the Census of Marine Life. Such collections ideally supplement traditional morphological voucher collections and are a major source of raw material fueling the integration of multiple subdisciplines in contemporary life science research. A major strength to an integrated approach exists in that descriptive taxonomy and molecular phylogenetic taxonomy together produce a synergy of resolution that neither can attain in isolation. The demand for specimens from GRCs now typically exceeds both the demand for traditional voucher specimens and the projection for the next decade is one of ever increasing use of GRCs. Thus, careful consideration should be made to sample genetic resources when the opportunity presents itself.

These collections and others like them face a unique set of challenges: how to balance the activities that grow, preserve, and promote their use, with consideration for maintaining optimal utility of the collection for future researchers. As this is a dynamic field of research and because opportunistic sampling is becoming increasingly difficult, it is incumbent upon the researcher to sample with “minimal regret” toward future needs. In other words, one should seek to preserve the broadest array of evidentiary value associated with a sample as possible, given the constraints of a particular expedition, funding, taxon, permitting, repository support, etc.

2. Collecting

Permits

For a thorough discussion of permitting issues and useful links, as well as many other topics of relevance to collecting tissue samples, see Prendini et al. (2002) and references therein (available on line: http://research.amnh.org/amcc/papers.html). Attention to permits should be initiated well in advance of any planned collecting activities where movement of biomaterials across international boundaries is concerned.

Methods

An adequate tissue sample will be dictated by the immediate needs of the research question and technique used to answer it, however to supplement individual research needs an archival specimen is always desirable and forms the basis of a synoptic GRC. Subsampling from existing collections is destructive and non-renewable without further collecting. Tissue culture methods have the advantage of providing an unlimited supply of genomic material but are labor-intensive to set up and culture conditions for most marine organisms have yet to be worked out. Those wishing to establish cultures should consult the primary literature or the specialists in the field (e.g., American Type Culture Collection).

Researchers planning to deposit archival specimens in a reference collection should consult the collection manager prior to undertaking their sampling trips to determine what containers are used at that particular institution and attempting to harmonize collecting efforts with the archival repository. This will ultimately reduce handling of time associated with specimen accession and thereby increase the quality of the specimen by minimizing the amount of time it has to be thawed, subsampled, and repackaged. Many frozen tissue collections store samples in standardized 2cc cryo vials (Prindini et al. 2002), and this can be taken as an adequate, albeit arbitrary optimal volume for an archival frozen tissue specimen. Consider using pre-labelled vials with a cryo-resistant label applied. The use of barcoded vials is now underway in some collections and some facilities will even provide specimen vials to legitimate collectors in advance of their collecting endeavors to facilitate specimen accession and retrieval. This system allows the collector to fill out data forms associated with each specimen and submit their data in a digital format which will help increase accession efficiency and minimize data transcription errors by repository staff.

Metadata

As collections move toward standardization for integrated information retrieval, collectors must be aware of current trends in data collection associated with biodiversity collections and strive to obtain as much relevant data as possible in association with their collections. Modern bioinformatics initiatives can link tissue specimen collection records with bibliographic citations, competing taxonomic determinations, geospatial referencing information, and much more. Collectors are advised to consult emerging data standards when considering what information to collect and how to format it before undertaking any collecting. For information on data exchange formats consult the Global Biodiversity Information Facility website (see: http://www.gbif.org/links/standards) and the Ocean Biogeographic Information System schema (http://www.iobis.org/technical.shtml).

Photography

Modern genetic resource collections strive to maintain a server with an online catalog for the resource, the most elaborate of which will host digital images illustrating various aspects of organismal morphology from whence the GRCs are derived. Baker and Monk (2001) have termed the use of digital imagery in natural history collections "eVouchers" and provide substantial justification for the utility and hence inclusion of this additional data layer in modern collections. Obvious considerations for generating useful eVouchers include references to scale and color calibration. While imaging dorsal, lateral and ventral views are critical, knowledge of the diagnostic features of a particular taxon facilitate capture of additional, more detailed photographs that are often critical for identification purposes.

3. Preserving

Researchers utilizing techniques such as BAC library construction (which requires very high molecular weight DNA) or microarrays and expressed-sequence-tag (EST) surveys of gene expression (which require intact RNA transcripts) cannot make use of most existing GRCs because the DNA and RNA have not been stored in the appropriate manner. With this in mind it is imperative that the method of preservation, both in the field and in the GRC itself, maximize the potential uses of the tissue, especially as specialized techniques in genomics become more taxonomically widespread (Couzins 2002). Storage of tissue in lysis buffer (Seutin et al. 1991) has the advantage of not requiring deep freezing and is very effective for isolating high molecular weight DNA, but by lysing cells it makes isolation of RNA or even of purified mtDNA a problem. Some protocols and storage buffers offer the ability to preserve RNA for PCR assays (Miller and Lambert 2003). However, even nitrogen storage will be inadequate for many molecular protocols if the tissues are left at ambient temperature for hours after collection. It would be terribly shortsighted to jeopardize the ability to retrieve transcriptomes and proteomes because of a limited attitude about storage of tissues.

Thus, collections made in association with the CoML should preserve as much of the biomolecular integrity of a specimen as possible when making tissue collections in association with the morphological voucher specimens, both of which should be deposited in publicly accessibly institutions dedicated to the long term care and support of such materials. Flash-freezing fresh tissue in liquid nitrogen, although logistically somewhat more complicated than fluid preservation, still represents the gold standard for preservation of tissues in the field (Engstrom, et al. 1999).

The current best practice in genetic resource collection involves a system of redundancy: in an ideal situation, two archival quality tissue samples will be immediately collected from each specimen, one frozen to preserve the broadest array of molecular characters possible and one placed into a preservation fluid, such as ethanol, to serve as a back up in case of a meltdown or loss of the frozen specimen. Taking this a step further, remote or off-site storage of one of the samples is also a recommended best practice by the International Society for Biological and Environmental Repositories (Pitt et al., 2005). Thus, CoML archival collections should consider designating a failsafe repository for storage of archival frozen tissue specimens.

This paradigm of hierarchical tissue preservation is ideal for multiple end uses of CoML collections. For example, the broad scale screening of standardized genotypic descriptors recommended via “DNA barcoding”(Hebert et al 2003), can utilize a sub-sample of the lower quality, fluid-preserved tissue sample; leaving the high quality frozen sample for future analysis involving genetic screens, expression studies, etc. This is imperative, as labile RNA species are sensitive to repeated thawing and freezing as involved with sub-sampling for prior DNA analyses.

For those who can collect flash frozen samples in the field, collecting multiple tissue types from rare or otherwise interesting focal taxa will facilitate subsequent studies in comparative gene expression and proteomics. Ultimately, the level of reductionism in sampling will be constrained by the research question being addressed and more pragmatic constraints such as budget and space available for multiple samples from the same organism. Such considerations should not be dismissed out of hand however, as the window of sampling opportunity for much of the world’s biodiversity is rapidly diminishing and the samples collected today might represent the only scientific evidence obtained from many species.

References

Couzins, J. 2002. NSF's ark draws alligators, algae and wasps. Science 297: 1638-1639.

Cook, J.A., G.H. Jarrell, A.M. Runck and J.R. Demboski. 1999. The Alaska Frozen Tissue Collection and associated electronic database: a resource for marine biotechnology. OCS Study MMS 99-0008. University of Alaska, Fairbanks.

Engstrom, M.D., R.W. Murphy and O. Haddrath. 1999. Sampling vertebrate collections for molecular research: practice and policies. pp 315-330 in D. Metsger and S. Byers (eds). Managing the Modern Herbarium: an Interdisciplinary Approach. Elton-Wolf, Vancouver.

Hebert, P.D.N., A. Cywinska, S.L. Ball and J. R. deWaard. 2002. Biological identifications through DNA barcodes. Proceedings of the Royal Society of London B: 270, 313–322. (DOI 10.1098/rspb.2002.2218.)

Miller, H. C., and D. M. Lambert. 2003. An evaluation of methods of blood preservation for RTPCR from endangered species. Conservation Genetics 4:651-654.

Monk, R.R. and R.J. Baker. 2001. eVouchers and the use of digital imagery in natural history collections. Museology, Museum of Texas Tech University. 10: 1-8.

International Society for Biological and Environmental Repositories (2005). Best Practices For Repositories. Cell Preservation Technology [In Press].

Prindini, L., R. Hanner and R. Desalle. 2002. Obtaining, storing and archiving specimens for molecular genetic research. in R. Desalle, G. Giribet and W. Wheeler (eds.), Methods and Tools in Biosciences and Medicine (MTBM) Techniques in Molecular Systematics and Evolution. Birkhauser, Basel.

Seutin, G., B. N. White and P. T. Boag. 1991 Preservation of avian blood and tissue samples for DNA analysis. Canadian Journal of Zoology 69, 82-90.