AgBio Sample Prep Technical Guide

Table of Contents

Letter from the Editor
Q1: How do you optimize the extraction and isolation of your sample while also minimizing contamination?
Q2: How do you develop or adapting existing protocols and validate them for your organisms of study?
Q3: How do you design appropriate primers or tags for your studies?
Q4: What steps do you take to reduce cost without sacrificing quality?
Q5: What challenges does working with polyploidy plants pose in sample preparation?
Q6: How else do you ensure good quality samples?
Agricultural Biology Grants
List of Resources

Download the PDF version here

Letter from the Editor

For any researcher, getting a good sample to analyze takes skill, perseverance, and a dash of good luck. But some researchers face greater challenges than others do. Scientists in the agricultural biology space focus on organisms as varied as Canola, soybeans, swine, wheat, and more — species whose genetic makeup isn't as accessible, nor as easy to work with as Arabidopsis, Drosophila, or mice. In this installment of Genome Technology's technical guide series, the University of Alberta's Nat Kav and his doctoral student William Yajima, along with Pat Heslop-Harrison tackle sample preparation issues due to the particular challenges of working with plants and phytopathogenic fungi, incomplete genomes, and a dearth of protocols. They give tips on how to ensure good quality samples and minimize contamination while keeping costs down — something that may well be on a lot of minds in these economic times. Cash-strapped researchers looking for more grant money should be sure to look at the list of available and recently funded grants to see what's out there.

As always, don't forget to check out the resources page for more information when you get stumped by a sample preparation problem. Special thanks to Pat Heslop-Harrison, Nat Kav, and William Yajima for taking the time to contribute to this technical guide.

— Ciara Curtin

Q1: How do you optimize the extraction and isolation of your sample while also minimizing contamination?

We need two types of sample for our molecular cytogenetics research program: purified genomic DNA for PCR analysis including diversity studies and gene isolation; and metaphase chromosome preparations for in situ hybridization to understand genome relationships, polyploidy and chromosome behavior. Optimization and minimizing contamination starts before we get the material: what species is it, where was it obtained, has it come from healthy plants? Where appropriate, we try to use material from international germplasm collections where the curators know the material well, have detailed records, verified the identification, and other scientists (and hopefully breeders exploiting our research in the future) will be able to access the same germplasm. We try to check species identification when we grow them, but could never grow 50 banana accessions to flowering. For both chromosomes and DNA, growing healthy plants is important. For chromosome preparations, we use the tips of actively growing, young roots. Getting root tips with many divisions and healthy nuclei is critical before starting an experiment that will take a couple of weeks. We routinely throw away two-thirds of fixation batches because there are not enough good divisions. With seeds, the first emerging root tips appear two days to a week after hydration on filter paper and these are used for preparations. The water used for seed germination is very important: our seeds get bottled drinking water, since distilled water is potentially acidic and has no minerals for the seeds, while tap water in labs can have heavy metals or plastics from plumbing systems and may have been in roof tanks for long periods. For many plants where seeds are not possible, or where we have only one plant, we use roots which grow against the side of the pot: the plant is re-potted about 10 days before we need the roots, then vigorous roots appear within this period. To get roots from trees, for example oil palm, in the field, we can scrape the soil surface, cover with a few centimeters of leaf mulch which is kept moist, and then find healthy roots growing up into the mulch after a few weeks.

— Pat Heslop-Harrison

The Kav laboratory performs extensive two-dimensional gel electrophoresis-based proteome-level investigations. We have extracted proteins from various plants as well as from phytopathogenic fungi and our approach involves the use of established protein extraction procedures described in the scientific literature as well as commercially available kits that have proven to be effective. If necessary, these procedures are modified to allow for optimal protein extraction from a particular tissue or organism. We have observed that different organisms present different challenges when attempting to extract proteins for 2D gel electrophoresis studies. For example, the presence of the highly abundant protein, Rubisco, in plants may mask the presence of other less abundant proteins in 2D gels. To avoid this, the fractionation of plant protein extracts and a subsequent 2D gel electrophoresis analysis might help.

We have also observed that extracting proteins from different tissues from the same organism might require the use of slightly different procedures. Root proteins and leaf proteins extracted using the same protocol produced 2D gel electrophoresis results that differed in image quality, with the leaf protein extract producing more distinct individual spots and less streaking of protein spots. This necessitated the modification of the protocol to efficiently extract root proteins that would generate high quality 2D gel results. Common modifications can involve the alteration of buffer composition (i.e. addition/deletion of components such as urea, thiourea, detergents, reducing agents, and other chaotropic agents). Additionally, when dealing with root samples and/or some plant tissue the presence of salt or phenolic compounds interferes with the isoelectric focusing step of 2D electrophoresis. The prior desalting of the samples using a commercially available desalting kit will significantly help in the separation of proteins isolated from such problem tissues. In general, when performing global proteome-level studies on tissue or an organism that we have not previously worked with, we first attempt to extract proteins using a TC A/acetone precipitation step as this should precipitate most proteins. This is followed by re-suspension of the proteins in a rehydration/sample buffer available from BioRad for use in 2D gel electrophoresis. Any modification to the buffers and protocol is based on the quality of the 2D gel images and is made on a case-by-case basis.

— Nat Kav & William Yajima

Q2: How do you develop or adapt existing protocols and validate them for your organisms of study?

With leaves for DNA isolation, we try to obtain enough quantity and use young leaves that are just reaching the fully expanded state. After this, they may accumulate more secondary products and pathogens. We prefer fresh leaves over dried or frozen. Where quarantine rules allow, we prefer to be sent fresh leaves and find the lack of handling, convenience of sending, and quality of extracted DNA is better than DNA from poorly preserved leaves. We start with our standard DNA extraction protocol with CT AB and find that, given enough quality leaf material, we usually obtain enough DNA for PCR and occasional Southern membranes. However, when the material is very limited or we extract low-quality DNA, then we move to use kits from the major molecular biology companies.

In making chromosome preparations, there are two very species-specific variables: the pretreatment required to synchronize and accumulate metaphases before fixation of the root, and the enzyme mixture/time used to digest the cell walls and spread the cells. Both need extensive optimization to get the best preparations. To accumulate metaphases, we treat excised root-tips in clean vials with aerated liquid to accumulate metaphases. We start with an 18 hour ice-water treatment for temperate species, particularly grasses, or 2 mM 8-hydroxyquinoline with dicotyledonous plants and species with small chromosomes for one to two hours at the plant growth temperature followed by one to two hours at 4°C. We also test plant response to water-saturated alpha-bromonaphthalene for two to six hours at growth temperature. It is important that the treatment temperature does not shock the roots, or few divisions will be seen.

— Pat Heslop-Harrison

Due to the inherent and unavoidable unpredictability involved in virtually all scientific research, oftentimes, adapting or modifying an existing protein extraction protocol for a new tissue sample and/or organism requires the adoption of a trial-and-error, case-by-case approach. Also, performing a step-by-step optimization of a protocol may be necessary to develop an effective protein extraction procedure. This may require the incorporation of many different procedures described by others to create one effective protocol. For example, in one study in which the cell wall proteome of a phytopathogenic fungus was investigated, it was necessary to first isolate the fungal cell wall. Prior to attempting any protein extraction, we searched the available scientific literature for an effective method to isolate the cell wall from the rest of the fungal cell. It was only after this was accomplished that we tried to extract proteins. The subsequent identification of proteins by mass spectrometry was performed.

— Nat Kav & William Yajima

Q3: How do you design appropriate primers or tags for your studies?

We are fortunate that some of the questions we ask use universal primers or probes, and the genomic DNA itself. We have universal primers which amplify various classes of retro-elements, the 5S and 45S rDNA sequences are similar enough across all plants that they can be used for in situ hybridization, and for looking at hybrids and polyploids we will use genomic DNA extracted from diploid ancestors directly as a probe. With PCR strategies for gene isolation (and sometimes retro-elements), primer design based on sequences from heterologous sequences takes a lot of effort, and we use various approaches of specific primers amplified at low annealing temperatures, degenerate primers at higher temperatures, and make sequential primers (mostly with a G/C clamp at the end), stepping along a few bases, in case there are mismatches. We usually run PCRs with single primers as well as the expected pairs, and clone the products before sequencing to see the primers and avoid mixed-product problems. When we are really frustrated with no products, we will re-amplify PCR products with the same or sometimes nested or hemi-nested primers. Finally, we make Southern transfers of both genomic DNA digests and PCR product, probing with heterologous genes to see if they are really there and our primer design needs refinement.

— Pat Heslop-Harrison

For the proteome-level studies typically performed in the Kav laboratory, it is usually not a prerequisite that the genome of the organism being studied is completely annotated. For the global proteomics-based studies of different plants and phytopathogenic fungi that we have undertaken, we have not required the labeling of proteins with any tags. However, the absence of a complete genome has been an issue when attempting to ascribe identities to proteins of interest using mass spectrometry. An incomplete genome in the various publicly available databases sometimes prevents the annotation of all of the identified proteins, which can limit the amount of information generated from a particular study.

— Nat Kav & William Yajima

Q4: What steps do you take to reduce cost without sacrificing quality?

The projects going on have different aspects of 'cost': well-funded consumables with limited labor, or people with time but limited consumables. And some come to gain training to go back to labs with minimal resources, so there would be no point in their developing, say, SSRs with multiplexed fluorochrome primers on an ABI when their project will only have a basic PCR and acrylamide gels. A key to all the projects though is good quality starting materials: healthy leaves and healthy plants. You can spend a lot of money and time using inadequate plant material whether for DNA or chromosomes.

— Pat Heslop-Harrison

Perhaps one of the most common methods used in the Kav laboratory to reduce costs without sacrificing quality involves optimizing protein extraction procedures using small-scale experiments. Rather than immediately attempting to extract proteins from multiple samples and/or large amounts of tissues, we typically perform protein extractions from one or two representative or control samples using a small amount of each tissue of interest. This allows us to minimize the amount of buffers and reagents that are required while simultaneously decreasing the time required to extract proteins. Once we are satisfied with the quality of the extracted protein sample, we will then perform a larger scale experiment. While we appreciate that scaling up does not merely involve the extrapolation of sample and buffer amounts, we usually find that performing small-scale experiments initially allows us to effectively optimize new protein extraction protocols. Similarly, when analyzing extracted proteins in SDS-PAGE gels, we typically run small (7 cm) gels before we use large (17 cm) gels, thereby reducing the associated costs of gel preparation and subsequent staining.

— Nat Kav & William Yajima

Q5: What challenges does working with polyploidy plants pose in sample preparation?

It is starting to look as though we can only fully define the polyploid nature and duplication present within genomes from full genomic sequencing. Few suspected the amount of duplication in Arabidopsis, or the results from papaya, until the sequencing was completed. So the prospect of $10,000 genome sequences will help a lot. It is extremely difficult to discover all copies of a gene in polyploids using PCR or hybridization strategies, and to say if a species is a polyploid and has duplications or not. A lot of our in situ hybridization work, using DNA from potential ancestors, is aimed at showing the derivation of polyploids, and which are autopolyploids with only one ancestral species, or hybrid-derived amphipolyploids.

— Pat Heslop-Harrison

The protein extraction protocols that are utilized are not dependent on the ploidy of the organism being studied.

— Nat Kav & William Yajima

Q6: How else do you ensure good quality samples?

I suppose my third important point, after healthy plants and healthy plants, is using healthy plants of known origin and verified genotype and species. If the material is not what you thought it was, then the work is entirely wasted. With less than optimum material, extracted DNA and chromosome preparations will be poor quality. It is important that growing the plants is not entirely delegated and the investigators are close to their plant material, not seeing it only as a white smudge in a tube or fluorescing chromosomes in the microscope.

— Pat Heslop-Harrison

Performing experiments using established standard operating procedures and employing good laboratory practices are paramount in ensuring consistent and reliable samples that will generate accurate and reproducible results. When performing proteome-level studies that involve the identification of proteins using mass spectrometry, it is important to be aware of common and easily-introduced contaminants that will adversely affect an experiment. Keratin from hair, skin cells, and fingernails can be inadvertently introduced into protein samples unless appropriate measures are taken, such as maintaining a clean work environment, using only clean/sterile lab supplies, and wearing gloves and a hairnet, if necessary. The improper storage of tissues and/or protein samples may also contribute to the generation of poor quality results. The degradation of proteins from inappropriate handling of samples can lead to inaccurate results and/or results that are not reproducible. Typically, plant and fungal samples that will be analyzed in our laboratory are used fresh or are harvested and flash-frozen in liquid nitrogen and then stored in the freezer if they are not used immediately. Furthermore, the repeated freezing and thawing of protein samples is always avoided in our laboratory in order to avoid protein degradation.

— Nat Kav & William Yajima

Agricultural Biology Grants

Grant Opportunities

Organization: U.S. Department of Energy and the U.S. Department of Agriculture
Award: $4 million for multiple awards
Details: The two US government agencies will be awarding funds for genomics-based research that will improve biomass and plant feedstock for fuel production, including ethanol. They are seeking applications for fundamental research to improve biomass or sustainability.
Contact: genomicsgtl.energy.gov

Organization: National Science Foundation
Award: Past awards have ranged between $20,000 to over $1 million
Details: This grant will support collaboration between US researchers and their counterparts in the developing world. Research should focus on agriculture, energy, or the environment. The call for applications says that "the technology must target crops grown locally in the developing countries and the traits that are most relevant to the local farmers and consumers."
Contact: www.nsf.gov

Organization: US Agency for International Development
Award: $2,200,000
Details: USAID is looking for applicants who will be taking a biotechnology-based approach to studying abiotic stress tolerant rice and wheat that can then be tested under field conditions in South East Asia, particularly in India. The most promising technology may be chosen for further development
Contact: www.grants.gov

Organization: National Science Foundation
Award: $16,000,000
Details: This award will fund investigators conducting basic research in plant genomics, particularly in plants of economic importance. The call for applications says that recent genomic advances using model organisms can now be applied to economically important plants, and that new and creative ideas are encouraged.
Contact: www.nsf.gov

Funded Grants


$30,827/ FY 2008
The function of small RNAs in the nitrogen response
Grantee: Gloria M. Coruzzi, New York University
Began Jan 1, 2008; Ends Dec 31, 2010
Coruzzi's long-term goal is to understand how nitrogen signaling controls N-assimilation, growth, and development. In the experiments funded by this grant she and her colleagues will explore, using microRNAs and other small RN As, how plants sense and respond to nitrogen at the molecular level.

$283,162/ FY 2008
Experimental annotation of the chicken genome
Grantee: Shane Burgess, Mississippi State
Began Jul 1, 2008; Ends Jun 30, 2012
With this grant, Burgess and his colleagues will be integrating high-throughput experimental data to comprehensively annotate the chicken genome, which was sequenced in 2004. They will also provide computational tools so that the community can access this information on their website.

$38,422/ FY 2008
Exploiting pathogen-induced cell death to create disease resistant plant
Grantee: Jean Greenberg, University of Chicago
Began Apr 10, 2008; Ends Mar 31, 2011
With this grant, Greenberg and her colleagues will be studying the Ralstonia solanacearum avirulence cell death effectors that activate defense responses in potatoes and will be identifying the plant's defense molecules that interact with those effectors.

List of resources

Here are some articles and websites to turn to when you have further questions.

Publications

Breseghello F, Sorrells ME. (2006). Association Mapping of Kernel Size and Milling Quality in Wheat (Triticum aestivum L.) Cultivars. Genetics. 172: 1165-1177.

Burnside J, Ouyang M, Anderson A, Bernberg E, Lu C, Meyers BC, Green PJ, Markis M, Isaacs G, Huang E, Morgan RW. (2008). Deep Sequencing of Chicken MicroRNAs. BMC Genomics. 9:185.

Jung K, Dardick C, Bartley LE, Cao P, Phetsom J, CanlasP, Seo YS, Shultz M, Ouyang S, Yuan Q, Frank BC, Ly E, Zheng Li, Jia Y, Hsia AP, An K, Chou HH, Rocke D, Lee GC, Schnable PS, An G, Buell CR, Ronald PC. (2008). Refinement of Light-Responsive Transcript Lists Using Rice Oligonucleotide Arrays: Evaluation of Gene-Redundancy. PLoS One. 3(10): e3337.

La Rota M, Kantety RV, Yu JK, Sorrells ME. (2005). Nonrandom distribution and frequencies of genomic and EST-derived microsatellite markers in rice, wheat, and barley. BMC Genomics. 6:23.

Paux E, Sourdille P, Salse J, Saintenac C, Choulet F, Leroy P, Korol A, Michalak M, Kianian S, Spielmeyer W, Lagudah E, Somers D, Kilian A, Alaux M, Vautrin S, Bergès H, Eversole K, Appels R, Safar J, Simkova H, Dolezel J, Bernard M, Feuillet C. A Physical Map of the 1-Gigabase Bread Wheat Chromosome 3B. Science. 322(5898): 101-104.

Ramakrishna W, Ma J, SanMiguel P, Emberton J, Dubcovsky J, Shiloff BA, Jiang Z, Rostoks N, Busso CS, Ogden M, Linton E, Kleinhofs A, Devos KM, Messing J, Bennetzen JL. (2002). Frequent Genic Rearrangements in Two Regions of Grass Genomes Identified by Comparative Sequence Analysis. Comp Funct Genomics. 3(2): 165–166.

Tyler BM, Tripathy S, Zhang X, Dehal P, Jiang RH Y, Aerts A, Arredondo FD, Baxter L, Bensasson D, Beynon JL, Chapman J, Damasceno CMB, Dorrance AE, Dou D, Dickerman AW, Dubchak IL, Garbelotto M, Gijzen M, Gordon SG, Govers F, Grunwald NJ, Huang W, Ivors KL, Jones RW, Kamoun S, Krampis K, Lamour KH, Lee MK, McDonald WH, Medina M, Meijer HJG, Nordberg EK, Maclean DJ, Ospina-Giraldo MD, Morris PF, Phuntumart V, Putnam NH, Rash S, Rose JKC, Sakihama Y, Salamov AA, Savidor A, Scheuring CF, Smith BM, Sobral BW S, Terry A, Torto-Alalibo TA, Win J, Xu Z, Zhang H, Grigoriev IV, Rokhsar DS, Boore JL. (2007). Phytophthora Genome Sequences Uncover Evolutionary Origins and Mechanisms of Pathogenesis. Science. 313(5791): 1261-1266.

Uauy C, Distelfeld A, Fahima T, Blechl A, Dubcovsky J. (2006). A NAC Gene Regulating Senescence Improves Grain Protein, Zinc, and Iron Content in Wheat. Science. 314(5803): 1298-1301.

Yu JK, Graznak E, Breseghello F, Tefera H, Sorrells ME. (2007). QTL mapping of agronomic traits in tef [Eragrostis tef (Zucc) Trotter]. BMC Plant Biology. 7:30.

Zhu W, Ouyang S, Iovene M, O'Brien K, Vuong H, Jiang J, Buell CR. (2008). Analysis of 90 Mb of the potato genome reveals conservation of gene structures and order with tomato but divergence in repetitive sequence composition. BMC Genomics. 9:286.

Websites

AgBase: www.agbase.msstate.edu
DNAAlignEditor Tool: http://maize.agron.missouri.edu/~hsanchez/DNAAlignment_Tool.html
MaizeMeister: www2.maizegenetics.net/bioinformatics/maizemeister/index.html
PowerMarker: statgen.ncsu.edu/powermarker
Polymorphism Between Two Accessions (Step One): www.panzea.org/db/searches/webform/polymorphic_between_accessions_step1