AgBio Sample Prep m e t h o d S

genome technology
AgBio
Sample Prep
a troubleshooting guide:
Experts share their tips on preparing
samples for agricultural biology
the genome web
intelligence network
m e t h o d s
Table of Contents
Letter from the Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Q1: How do you optimize the extraction and isolation of
your sample while also minimizing contamination? . . 5
Q2: How do you develop or adapting existing protocols
and validate them for your organisms of study?. . . . . . 8
Q3: How do you design appropriate primers or tags
for your studies?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Q4: What steps do you take to reduce cost without
sacrificing quality?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Q5: What challenges does working with polyploidy
plants pose in sample preparation?. . . . . . . . . . . . . . . . 11
Q6: How else do you ensure good quality samples?. . . . . 12
Agricultural Biology Grants. . . . . . . . . . . . . . . . . . . . . . . . . . . 13
List of Resources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
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
AgBio Sample Prep
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
December 2008/JANUARY 2009 genome technology 3
The GenomeWeb
Intelligence Network.
Connecting the dots for researchers worldwide.
How do you optimize the extraction
and isolation of your sample while
also minimizing contamination?
We need two types of sample
tion when we grow them, but
in labs can have heavy met-
for our molecular cytogenet-
could never grow 50 banana
als or plastics from plumbing
ics research program: puri-
accessions
flowering.
systems and may have been
fied genomic DNA for PCR
For both chromosomes and
in roof tanks for long periods.
analysis including diversity
DNA, growing healthy plants
For many plants where seeds
studies and gene isolation;
is important.
are not possible, or where we
and
For chromosome prep-
have only one plant, we use
some preparations for in
arations, we use the tips
roots which grow against the
situ hybridization to under-
of actively growing, young
side of the pot: the plant is
stand genome relationships,
roots. Getting root tips with
repotted about 10 days be-
polyploidy and chromosome
many divisions and healthy
fore we need the roots, then
behavior. Optimization and
nuclei is critical before start-
vigorous roots appear within
minimizing
contamination
ing an experiment that will
starts before we get the
take a couple of weeks. We
material: what species is it,
routinely throw away two-
where was it obtained, has
thirds of fixation batches be-
it come from healthy plants?
cause there are not enough
Where appropriate, we try to
good divisions. With seeds,
use material from interna-
the first emerging root tips
tional germplasm collections
appear two days to a week
where the curators know the
after
material well, have detailed
paper and these are used for
records, verified the identi-
preparations. The water used
fication, and other scientists
for seed germination is very
(and hopefully breeders ex-
important: our seeds get
ploiting our research in the
bottled drinking water, since
future) will be able to access
distilled water is potentially
the same germplasm. We try
acidic and has no minerals
“Where
appropriate,
we try to use
material from
international
germplasm
collections where
the curators know
the material well.”
to check species identifica-
for the seeds, while tap water
— Pat Heslop-Harrison
metaphase
AgBio Sample Prep
chromo-
to
hydration
on
filter
this period. To get roots from
trees, for example oil palm,
in the field, we can scrape
the soil surface, cover with
December 2008/JANUARY 2009 genome technology 5
addition/deletion
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
gel
two-dimensional
electrophoresis-based
proteome-level
investiga-
tions. We have extracted pro-
of
com-
“Extracting
proteins from
different tissues
from the same
organism might
require the use
of slightly different
procedures.”
ponents such as urea, thio-
— Nat Kav & William Yajima
of 2D electrophoresis. The
urea, 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
prior desalting of the sam-
teins from various plants as
ples using a commercially
well as from phytopathogen-
tracts and a subsequent 2D
ic fungi and our approach
gel electrophoresis analysis
involves the use of estab-
might help.
lished
extraction
We have also observed that
procedures described in the
extracting proteins from dif-
scientific literature as well
ferent tissues from the same
as commercially available
organism might require the
global proteome-level stud-
kits that have proven to be
use of slightly different pro-
ies on tissue or an organism
effective. If necessary, these
cedures. Root proteins and
that we have not previously
procedures
modified
leaf proteins extracted using
worked with, we first attempt
to allow for optimal protein
the same protocol produced
to extract proteins using a
extraction from a particular
2D gel electrophoresis re-
TCA/acetone
tissue or organism. We have
sults that differed in image
step as this should precipi-
observed that different or-
quality, with the leaf protein
tate most proteins. This is
ganisms present different
extract producing more dis-
followed by re-suspension
challenges when attempting
tinct individual spots and
of the proteins in a rehydra-
to extract proteins for 2D
less
tion/sample
gel electrophoresis studies.
spots. This necessitated the
able from BioRad for use in
For example, the presence
modification of the protocol
2D gel electrophoresis. Any
of the highly abundant pro-
to efficiently extract root
modification to the buffers
tein, Rubisco, in plants may
proteins that would generate
and protocol is based on the
mask the presence of other
high quality 2D gel results.
quality of the 2D gel images
less abundant proteins in 2D
Common
and is made on a case-by-
gels. To avoid this, the frac-
can involve the alteration
tionation of plant protein ex-
of buffer composition (i.e.
protein
are
6 TEch guide december 2008/JANUARY 2009
streaking
of
protein
modifications
available desalting kit will
significantly help in the separation of proteins isolated
from such problem tissues.
In general, when performing
precipitation
buffer
avail-
case basis.
— Nat Kav & William Yajima
AgBio Sample Prep
How do you develop or adapt
existing protocols and validate
them for your organisms of study?
With leaves for DNA isolation,
metaphases before fixation of
research, oftentimes, adapting
we try to obtain enough quanti-
the root, and the enzyme mix-
or modifying an existing protein
ty and use young leaves that are
ture/time used to digest the cell
extraction protocol for a new
just reaching the fully expanded
walls and spread the cells. Both
tissue sample and/or organ-
state. After this, they may ac-
need extensive optimization to
ism requires the adoption of
cumulate
secondary
get the best preparations. To
a trial-and-error, case-by-case
products and pathogens. We
accumulate metaphases, we
approach. Also, performing a
prefer fresh leaves over dried or
treat excised root-tips in clean
step-by-step optimization of
frozen. Where quarantine rules
vials with aerated liquid to ac-
a protocol may be necessary
allow, we prefer to be sent fresh
cumulate
to develop an effective pro-
leaves and find the lack of han-
start with an 18 hour ice-water
tein extraction procedure. This
dling, convenience of sending,
treatment for temperate spe-
may require the incorporation
and quality of extracted DNA
cies, particularly grasses, or 2
of many different procedures
is better than DNA from poorly
mM 8-hydroxyquinoline with
described by others to create
preserved leaves. We start with
dicotyledonous
and
one effective protocol. For ex-
our standard DNA extraction
species with small chromo-
ample, in one study in which
protocol with CTAB and find
somes for one to two hours at
the cell wall proteome of a phy-
that, given enough quality leaf
the plant growth temperature
topathogenic fungus was in-
material, we usually obtain
followed by one to two hours at
vestigated, it was necessary to
enough DNA for PCR and
4°C. We also test plant response
first isolate the fungal cell wall.
occasional
more
Southern
metaphases.
plants
We
mem-
to water-saturated alpha-bro-
Prior to attempting any protein
branes. However, when the
monaphthalene for two to six
extraction, we searched the
material is very limited or
hours at growth temperature. It
available scientific literature for
we extract low-quality DNA,
is important that the treatment
an effective method to isolate
then we move to use kits
temperature does not shock
the cell wall from the rest of the
from the major molecular
the roots, or few divisions will
fungal cell. It was only after
biology companies.
be seen.
this was accomplished that
In making chromosome
—Pat Heslop-Harrison
preparations, there are two
we tried to extract proteins.
The subsequent identifica-
very species-specific variables:
Due to the inherent and un-
tion of proteins by mass spec-
the pretreatment required to
avoidable unpredictability in-
trometry was performed.
synchronize and accumulate
volved in virtually all scientific
8 TEch guide december 2008/JANUARY 2009
— Nat Kav & William Yajima
AgBio Sample Prep
How do you design appropriate
primers or tags for your studies?
We are fortunate that some of
mismatches. We usually run
topathogenic fungi that we
the questions we ask use uni-
PCRs with single primers as
have undertaken, we have
versal primers or probes, and
well as the expected pairs,
not required the labeling
the genomic DNA itself. We
and clone the products be-
of proteins with any tags.
have universal primers which
fore sequencing to see the
However, the absence of a
amplify various classes of ret-
primers and avoid mixed-
complete genome has been
roelements, the 5S and 45S
product problems. When we
an issue when attempt-
rDNA sequences are similar
are really frustrated with no
ing to ascribe identities
enough across all plants that
products, we will reamplify
to proteins of interest us-
they can be used for in situ hy-
PCR products with the same
ing mass spectrometry. An
bridization, and for looking at
or sometimes nested or he-
incomplete genome in the
hybrids and polyploids we will
mi-nested primers. Finally,
various publicly available da-
we make Southern trans-
tabases sometimes prevents
fers of both genomic DNA
the annotation of all of the
digests and PCR product,
identified proteins, which
probing with heterologous
can limit the amount of
genes to see if they are really
information generated from a
there and our primer design
particular study.
use genomic DNA extracted
from diploid ancestors directly as a probe. With PCR strategies for gene isolation (and
sometimes
retroelements),
primer design based on sequences from heterologous
sequences takes a lot of effort,
— Nat Kav & William Yajima
needs refinement.
— Pat Heslop-Harrison
and we use various approach-
For the proteome-level stud-
es of specific primers ampli-
ies typically performed in
fied at low annealing temper-
the Kav laboratory, it is usu-
atures, degenerate primers
ally not a prerequisite that
at higher temperatures, and
the genome of the organism
make
primers
being studied is completely
(mostly with a G/C clamp at
annotated. For the global
the end), stepping along a
proteomics-based
few bases, in case there are
of different plants and phy-
sequential
AgBio Sample Prep
studies
“The 5S and 45S
rDNA sequences
are similar enough
across all plants
that they can be
used for in situ
hybridization.”
—Pat Heslop-Harrison
December 2008/JANUARY 2009 genome technology 9
What steps do you take
to reduce cost without
sacrificing quality?
The projects going on have
procedures
different aspects of ‘cost’:
scale experiments. Rather
analyzing
well-funded
consumables
than immediately attempt-
teins in SDS-PAGE gels, we
with limited labor, or people
ing to extract proteins from
typically run small (7 cm)
with time but limited con-
multiple
sumables. And some come
large amounts of tissues,
(17
to gain training to go back
we typically perform pro-
reducing
to labs with minimal re-
tein extractions from one
costs of gel preparation and
sources, so there would be
or two representative or
subsequent staining.
no point in their developing,
control samples using a
say, SSRs with multiplexed
small amount of each tis-
fluorochrome primers on an
sue of interest. This allows
ABI when their project will
us to minimize the amount
only have a basic PCR and
of
acrylamide gels. A key to all
that are required while si-
the projects though is good
multaneously
quality starting materials:
the time required to extract
healthy leaves and healthy
proteins. Once we are satis-
plants. You can spend a lot of
fied with the quality of the
money and time using inade-
extracted protein sample,
quate plant material whether
we will then perform a larger
for DNA or chromosomes.
scale experiment. While we
— Pat Heslop-Harrison
using
samples
buffers
and
small-
and/or
reagents
decreasing
appreciate that scaling up
does not merely involve the
Perhaps one of the most
extrapolation of sample and
common methods used in
buffer amounts, we usually
the Kav laboratory to re-
find that performing small-
duce costs without sacri-
scale experiments initially
ficing quality involves op-
allows us to effectively opti-
timizing protein extraction
mize new protein extraction
10 TEch guide december 2008/january 2009
protocols. Similarly, when
extracted
pro-
gels before we use large
cm)
gels,
the
thereby
associated
— Nat Kav & William Yajima
“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.”
—Nat Kav & William Yajima
AgBio Sample Prep
What challenges does working
with polyploidy plants pose in
sample preparation?
It is starting to look as
genome sequences will help
the derivation of polyploids,
though we can only fully de-
a lot. It is extremely diffi-
and which are autopoly-
fine the polyploid nature and
cult to discover all copies of
ploids with only one ances-
duplication present within
a gene in polyploids using
tral species, or hybrid-de-
genomes from full genomic
PCR or hybridization strate-
sequencing. Few suspected
gies, and to say if a species
the amount of duplication
is a polyploid and has dupli-
The protein extraction proto-
in Arabidopsis, or the re-
cations or not. A lot of our in
cols that are utilized are not
sults from papaya, until the
situ hybridization work, us-
dependent on the ploidy of
sequencing was completed.
ing DNA from potential an-
the organism being studied.
So the prospect of $10,000
cestors, is aimed at showing
rived amphipolyploids.
— Pat Heslop-Harrison
— Nat Kav & William Yajima
How else do you ensure
good quality samples?
I suppose my third impor-
generate accurate and re-
sults and/or results that
tant point, after healthy
producible
results. When
are not reproducible. Typi-
plants and healthy plants,
performing proteome-level
cally, plant and fungal sam-
is using healthy plants of
studies
the
ples that will be analyzed
known origin and verified
identification
proteins
in our laboratory are used
genotype and species. If
using mass spectrometry, it
fresh or are harvested and
the material is not what
is important to be aware of
you thought it was, then
flash-frozen in liquid nitro-
common and easily-intro-
the work is entirely wasted.
gen and then stored in the
duced contaminants that
With less than optimum ma-
freezer if they are not used
will adversely affect an ex-
terial, extracted DNA and
immediately. Furthermore,
periment. Keratin from hair,
chromosome preparations
the repeated freezing and
skin cells, and fingernails can
will be poor quality. It is
be inadvertently introduced
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
that
involve
of
into protein samples unless
appropriate measures are
taken, such as maintaining
a clean work environment,
using
lab
only
supplies,
clean/sterile
and
wear-
ing gloves and a hairnet,
if necessary.
The improper storage of
experiments
tissues and/or protein sam-
using established standard
ples may also contribute to
operating procedures and
the generation of poor qual-
employing good laboratory
ity results. The degradation
practices
paramount
of proteins from inappro-
in ensuring consistent and
priate handling of samples
reliable samples that will
can lead to inaccurate re-
Performing
are
12 TEch guide december 2008/JANUARY 2009
thawing of protein samples
is always avoided in our
laboratory in order to avoid
protein degradation.
— Nat Kav & William Yajima
“The repeated
freezing and
thawing of
protein samples
is always avoided
in our laboratory
in order to
avoid protein
degradation.”
—Nat Kav & William Yajima
AgBio Sample Prep
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 impor-
AgBio Sample Prep
tant 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 Nassimilation, growth, and development.
In the experiments funded by this grant
she and her colleagues will explore, using
microRNAs and other small RNAs, how
plants sense and respond to nitogen 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.
December 2008/JANUARY 2009 genome technology 13
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
RHY, Aerts A, Arredondo FD, Baxter L, Bensasson D, Beynon JL, Chapman J, Damasceno
14 TEch guide december 2008/JANUARY 2009
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 BWS, 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
AgBio Sample Prep