Table of contents
Letter from the Editor . . . . . . . . . . . . . . . . . . . . . . .5
Index of Experts . . . . . . . . . . . . . . . . . . . . . . . . . . .6
Q1: Which sample collection and storage
techniques yield optimal RNA? . . . . . . . . . . .7
Q2: What techniques do you use to assess
and isolate high-quality RNA? . . . . . . . . . . . .9
Q3: How do you establish controls for
heterogeneous or difficult samples? . . . . . . . .10
Q4: How do you improve hybridization
specificity and sensitivity? . . . . . . . . . . . . .11
Q5: What tips do you have for clean-up
and handling of arrays? . . . . . . . . . . . . . . . .12
List of Resources . . . . . . . . . . . . . . . . . . . . . . . . . .14
Genome Technology
Microarray Sample Prep Tech Guide
3
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Letter from the editor
Welcome to the latest installment in
Genome Technology’s reference guide
series. In this second issue, we are
pleased to present expert insights on
optimizing sample prep for microarray
experiments.
Sample prep can make or break
your microarray experiment. Whether your samples
come from the clinic or the bench, the tips compiled in
the following pages will help you maximize the quality
of your arrays.
Since making their debut, microarrays have gained
popularity at a remarkable pace. Commercial chips
have found their way into labs everywhere, luring
investigators with the promise of generating reliable
data on thousands of genes at once. The tool will no
doubt continue to reach new users as the costs
involved in setting up and running a microarray
experiment keep dropping, while dedicated core
facilities spring up in both universities and industry.
Genome Technology
erie-genome-half-h.indd 1
Microarray technology may be widely available,
but important issues remain. From sample selection to
data analysis across platforms, groups are working to
agree on a number of quality control guidelines for all
steps in the process. Only in this way can microarrays go
from standard tool to standardized technology.
One of the most critical steps in the process is
sample preparation. Accurate and meaningful data is
highly dependent on the quality of isolated nucleic acid
or protein samples, and these, in turn, are contingent
on correct acquisition, extraction, and purification
procedures. Learning how to master correct handling
of samples takes experience, which can be costly to
attain (in both reagents and time).
The microarray experts on our panel offer detailed
advice on issues in sample prep. Keep this guide on
hand for quick answers to common problems
concerning sample collection, isolation, establishing
controls, and more.
— Jennifer Crebs
Microarray Sample Prep Tech Guide
5
12/9/05 10:43:20 AM
Index of experts
Genome Technology would like to thank the following
contributors for taking the time to respond to the
questions in this tech guide:
Shoshi Kikuchi
Head of the Laboratory of
Gene Expression
Department of Molecular Genetics
National Institute of Agrobiological
Sciences, Japan
Philippe Demougin
Biozentrum
University of Basel
André Ponton
Director, Microarray Laboratory
McGill University and
Genome Quebec Innovation Centre
Gary Hardiman
Director, BioMedical Genomics
Microarray Facility
Assistant Professor, Department
of Medicine
University of California, San Diego
Scott Tenenbaum
Assistant Professor,
University of Albany-SUNY
GEN*NY*SIS Center for Excellence
in Cancer Genomics
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the business day.
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6
Microarray Sample Prep Tech Guide
Genome Technology
Which sample collection
and storage techniques
yield optimal RNA?
contact with RNA. So in this case tissues must be
The key parameter for yielding optimal total RNA
homogenized (in a solution inhibiting RNase activity)
quality is to immobilize tissues as fast as possible
very rapidly going from a frozen stage (transport
after sampling. Proceeding fast is crucial because it
samples on dry ice) to a completely homogenized
doesn’t let time for RNA level of expression to
stage within seconds.
change because of technical stress, and more
— Philippe Demougin
importantly, RNases won’t have time to get in
contact with RNA molecules.
I have found that both solid
Tissues can be fetched into
liquid nitrogen or in RNAlater
“Solid phase methods work phase and chaotropic salt
methods give adequate results.
(Ambion, Cat# 7020). Either
very well in that they The advantage with chaotropic
way is good but RNAlater
requires the samples to be small
generate very clean RNA, but salt methods is that once
released from the cells or
so that the solution can get into
there is greater risk of tissues, the RNA is less prone to
the tissue and protect RNA.
initial degradation due to the
RNAlater is preferred over liquid
degradation.”
inhibitory effects of guanadium
nitrogen especially in two
— Gary Hardiman
and phenol. These can later be
cases: in surgery room where
a problem as they can interfere
the use of liquid nitrogen is
with the reverse transcription
commonly prohibited and in
reaction by inhibiting the activity of the reverse
case further dissection steps are required for
transcriptase enzyme.
isolating a tiny part of tissue (embryo
Often I see RNA that is structurally intact but
microdissection for instance).
cannot be converted into high-quality target material
Tissues are left in RNAlater overnight at 4°C,
due to the presence of contaminants. Ethanol
then stored at -20°C or -70°C. If snap-frozen in
precipitation or clean-up with a solid phase method
liquid nitrogen, they are stored at -70°C following
is required. Solid phase methods work very well in
snap freezing. Proceeding to tissue isolation, the use
that they generate very clean RNA, but there is
of RNAlater allows more flexibility. Since the solution
greater risk of degradation. This means that an
penetrated the tissue before storage, samples can be
RNase inhibitor must be used. Additionally, the yields
thawed on ice and tissue homogenization is
of RNA are generally lower.
performed with no rush.
— Gary Hardiman
For tissues stored at -70°C after snap freezing in
liquid nitrogen, the situation is more critical: even
We are preparing total RNA from plant tissue. For the
partial or short thawing can result in dramatic RNA
microarray analysis and
degradation because the tissues are destructurized
(continued on page 13)
RNA preparation, to
by the temperature shock and RNases will be in
Genome Technology
Microarray Sample Prep Tech Guide
7
What techniques do you
use to assess and isolate
high-quality RNA?
Whatever the tissue from which RNA is isolated, we
systematically pass total RNA over a silica-based
column (e.g. Qiagen RNeasy) in order to clean it up
from genomic DNA, salts, phenol… We routinely
used a Qiagen TissueLyser apparatus (Cat# 85220)
for homogenizing the tissues. Tissue is [shaken] at
high frequency in a tube in the presence of a single
5mm metal bead.
The protocol applied depends on how difficult it
is to disrupt the tissue and open up the cells: if the
moderate lysis power of the lysis solution (RLT Buffer)
provided with the RNeasy kit is sufficient,
homogenate is directly passed over the silica
columns. This is typically the case for cell culture or
soft tissues. But a phenol-based chemical (e.g.
TRIzol) is required for most tissues either because
they are tougher to break up, richer in proteins, or
richer in lipids.
One can adjust the time and frequency of
homogenization. 25Hz for two minutes is a safe
starting point. One should always homogenize
the minimum time necessary for a complete
homogenization.
— Philippe Demougin
High-quality RNA can be isolated using either
chaotropic salt and solid phase methods, or a
combination of the two. The RNA can be assessed by
spectrometry and gel electrophoresis if quantities
permit. The ratio of the absorbance readings at 260
and 280 should be between 1.8 and 2. Typically the
ratio of high to low molecular weight ribosomal RNA
should be about 2.
DEPC-treated water should be avoided for resuspending RNA as this can inhibit enzymatic
Genome Technology
reactions. Additionally, if there is a need to
concentrate the RNA, this should be done by salt
precipitation or desiccation with a low heat setting.
Once RNA has been dried to completion, it can be
very hard or impossible to re-suspend.
Tissue provides much more of a challenge for
RNA extraction than cell lines. Typically, the material
needs to be well homogenized prior to extraction.
When using a chaotropic salt method, it is critical to
avoid organic phase contamination of the RNA
supernatant. After centrifugation, one should avoid
taking material near the aqueous organic interphase
as this is a source of genomic DNA contamination.
Agilent Bioanalyzer analysis is very useful in
assessing the integrity of total RNA. The 28 and 18S
ribosomal RNA species should be present as strong
peaks. There should be an absence of low molecular
weight products (indicating degradation) and high
molecular weight contaminating genomic DNA.
— Gary Hardiman
[In order to homogenize plant tissue prior to RNA
extraction] we are using Multi-Beads Shocker (Yasui
Kikai). This product is the most important tool for the
preparation of RNA or DNA from hard plant tissues.
Many researchers in the plant field out[side] of Japan
want to purchase this tool, but [it] is only purchasable
in Japan.
— Shoshi Kikuchi
We received all kind of RNA from different tissues and
species. In general TRIzol extraction followed by
Qiagen column clean-up give good results.
— André Ponton
Microarray Sample Prep Tech Guide
9
How do you establish
controls for heterogeneous
or difficult samples?
wavelengths) using a NanoDrop spectrophotometer
A mini-electrophoresis system such as the Agilent
will reveal the purity of the RNA and the presence of
Bioanalyzer or the Bio-Rad Experion allows fast
DNA, proteins, or carbohydrates. The integrity of the
analysis of RNA samples. Most importantly, they
RNA can be assessed using an Agilent Bioanalyzer
allow a precise evaluation of their quality. The
and compared to control RNA samples.
integrity of 28S and 18S ribosomal RNA is used to
— Gary Hardiman
assess the quality of the total RNA sample. The
sharper the peaks, the better the quality. A unique
We are measuring the amount of RNA by [using]
feature of the Bioanalyzer is the scoring (from 0 to
NanoDrop OD measurement equipment and by the
10) of the total RNA quality. For the first time
Bioanalyzer system, [by which] degradation of RNA
researchers have a tool for assessing the RNA quality
samples is monitored.
in an objective manner. RNA quality is very much
— Shoshi Kikuchi
dependent of the tissue, some tissues being naturally
richer in RNases than others. By
“You have to do a lot of We use the same standard
experience we simply know
that most people use.
what pattern and score to get
repeats … our standard is controls
Using bacterial spikes and
for various tissues. For difficult
three biological replicates, things like that, one can
samples, measures are taken for
one sample from the
shortening the times, increasing
each technically replicated average
next by gauging off of those
the homogenization force if
twice.”
— Scott Tenenbaum
types of controls. But I find
necessary, working on ice, and
those to be only partly useful.
working faster. Getting things
They tend to control for certain aspects of the
organized in order to work faster helps a lot.
technique but not the whole method. For instance,
A large amount of contaminated genomic DNA
the bacterial spikes will control for labeling and
in an RNA sample can lead to unexpected migration
hybridization issues, but not really for earlier RNA
profiles and misleading interpretation.
quality. There’s no way to control for that, because
— Philippe Demougin
you’re adding the control a step after the process
occurred.
The most difficult samples I have found for
You have to do a lot of repeats. I’m funded
microarray experimentation are those derived from
through the ENCODE project, which is part of the
laser capture experiments, where the yields are low
NIH and the National Human Genome Research
and there may be contaminating genomic DNA
Institute, and our standard is three biological
present. The extracted RNA should be compared to a
replicates, each of which is technically replicated
control — for example, the Stratagene or Clontech
twice. If you do literally six
reference RNA. If sufficient RNA is available, optical
repeats on something, an (continued on page 13)
density readings at 260nm and 280nm (and other
10
Microarray Sample Prep Tech Guide
Genome Technology
How do you improve
hybridization specificity
and sensitivity?
We hybridized only Affymetrix arrays. Procedures are
very much standardized, and we stick to the default
recommendations. Reliable arrays and [automated]
procedures being the key advantages of the
GeneChips versus spotted arrays.
— Philippe Demougin
For the various commercial catalog arrays, I usually
adhere to standard operating protocols, as much of
this has been worked out and optimized by the array
manufacturer — deviating from the SOPs can mean
you are on your own if the experiment fails.
For homemade or boutique arrays where
optimization is usually required, several components
can be adjusted to improve hybridization specificity
and sensitivity. Firstly, good oligonucleotide design is
key. The investigator needs to ensure that the oligos
are specific to the mRNA of interest, and design probes
that do not contain fold-back loops. Hybridization
conditions are optimized depending on the slide
surface employed; sometimes a pre-hybridization step
may improve the specificity. The composition of
hybridization buffers is important. I favor formamidebased buffers as you can reduce the temperature and
maintain hybridization specificity. One of the concerns
with aqueous buffers at higher temperatures is the
fear of drying out the hybridization solution.
As regards sensitivity, improvements are
generally seen when you move from static to agitated
hybridization. The use of a reciprocal shaker at a low
speed setting ensures a wave-like motion and
prevents localized target depletion. The ideal is an
automated hybridization/fluidic station that ensures
consistent hybridization and maximum sensitivity.
— Gary Hardiman
Genome Technology
In our case, we are using the oligoarray system
produced by Agilent Technologies. We just follow
the recommended protocols from them.
— Shoshi Kikuchi
This is not a concern with Affymetrix microarrays as
long as the sample quality is very good.
— André Ponton
That’s really a platform question. Affymetrix has
made great strides in the last several years to clear up
issues with their platform and get around the
problems. For instance, there are only 25 base
probes, but the 11 probes they now use on average
per gene are very well selected. Some of the longer
probe-based arrays do a better job, and NimbleGen
and GE have some interesting products. For the
longer 50-60mer probes, the signal to noise is better.
But there are issues there too.
All [platforms] inherently have a level of noise
that I think most traditionally trained, reductionist
biologists have to get comfortable with. There are a
lot of variables, fairly involved methods, and there’s
a degree of fluctuation that is inherent in the system.
Some researchers have a lot of issues with that; they
just don’t like that things are bouncing around a lot.
The simplest solution I’ve had for that is I almost
always go for the low-hanging fruit: I usually use a
fourfold cutoff or greater and it cleans it up pretty
quick. It’s one of the nice things about working on
genomic-scale stuff — there are usually so many
things to look for that it’s OK if you keep your
stringency pretty high, because lots and lots of things
will still meet that criterion.
— Scott Tenenbaum
Microarray Sample Prep Tech Guide
11
What tips do you have for
clean-up and handling of
arrays?
A general precaution when dealing with fluorescent
detection of arrays is to avoid getting any particles
(which are usually highly autofluorescent) in contact
with the arrays. Work in a dust-free environment;
use only powder-free gloves; filter all solutions
(0.22µm), and make sure they don’t get
contaminated (discard any turbid solution);
use double distilled water and eventually DEPCtreated. Centrifuge the probe before loading the
upper part of it onto the arrays, especially if a
column-based system was used upstream during the
synthesis. Particles from the column are likely to be
autofluorescent.
— Philippe Demougin
The investigator should avoid touching the
arrays and avoid using powdered gloves, which
often create fluorescent artifacts. A clean lab or
dust-free area is essential. When washing arrays, the
arrays should be fully submerged or edge effects will
be seen. When drying arrays, I favor centrifugation
over use of nitrogen. It’s also not a good idea to
process too many arrays at once, as this can lead to
noisy arrays. Arrays should be kept separate from
each other. By this I mean not exactly side by side on
a washing tray. It’s not a good idea to have
fluorescent target being washed off one array and
binding non-specifically to an adjacent array that is
also being washed.
— Gary Hardiman
Normally [by] spraying the N2 gas.
— Shoshi Kikuchi
12
In case of Affymetrix,
recommendations.
we
follow
their
— André Ponton
If you’re getting in on the field and it’s new to you, I
would strongly encourage you [to] work with a core
facility that has a lot of experience doing it. Core
facilities that run one or two microarrays per month
are really new at it themselves. You’d be better off
working with people that run hundreds of arrays on
a regular basis. They are much better at doing the
technique and the analysis. I think people who spend
a lot of time analyzing microarray data are much
better suited to work with when you’re getting into
this field.
I have too often seen researchers who finally get
enough money to run some arrays, but once they
get their data back, they’re really on their own. They
just don’t know what to do with the volumes of data
one gets. There’s a little bit of hand holding, but not
really enough. And so they don’t make any headway
on what could have been some pretty good data.
They spend a couple of months scratching their head
and they eventually do exactly what I think they
shouldn’t do, and that is: they take a few genes
they’re interested in, and say, ‘OK, that gene I know
about, so I’ll go focus on that.’ It defeats the whole
purpose of doing this big genomic-scale analysis if
you’re going to look at 30,000 genes and throw
them all out to look at one. It seems kind of foolish.
So, that would be my big suggestion: Get with a
group that can give you the analysis support as well
as run a lot of arrays, so they know what’s going on.
— Scott Tenenbaum
Microarray Sample Prep Tech Guide
Genome Technology
Q1: Which sample collection and
storage techniques yield
optimal RNA? (continued from p.7)
avoid the noise of gene expression by cutting, etc.,
we normally prepare liquid nitrogen before tissue
preparation. Then whole plant (control or stresstreated plants) will be frozen in liquid nitrogen and,
after completely frozen, [the] RNA preparation
process will be started. This kind of treatment can
avoid unexpected gene expression during sample
preparation process.
As for the capture of specific tissue — such as
the specific cell layer, specific organ — we will capture
the
tissue
at
normal
temperature
and,
after capture, tissue will be frozen in liquid nitrogen.
— Shoshi Kikuchi
Usually it is best to keep the RNA in RNase-free water
(commercial source) and put aliquots at -80°C. It is
best to avoid multiple freeze-thaw cycles.
— André Ponton
Usually, we use Qiagen’s RNeasy prep or TRIzol
extractions. I store my RNA in ethanol at -80°C. In my
experience, you either have an RNase problem or you
don’t. If you don’t, it’s pretty stable. And if you do, it
doesn’t matter what you do — it’s gone. The trick is
to not have any RNase problems. My RNAs are usually
stable, especially at -80°C in ethanol, they’re good
for a year, no problem.
— Scott Tenenbaum
Q3: How do you establish controls
for heterogeneous or difficult
samples? (continued from p.10)
outlier becomes pretty obviously an outlier. Then you
can either selectively remove it or lessen its impact.
I think the biggest criticism of the field is most
researchers will err on the side of doing more time
points, rather than doing fewer time points and more
replicates. So they’ll do one sample each at six
different time points, as opposed to doing three
replicates each at two time points. The data in the
end is much more believable that way.
— Scott Tenenbaum
Genome Technology
Microarray Sample Prep Tech Guide
13
List of resources
Our microarray experts referred to a number of
products, which we’ve compiled below. The
recommended publications feature even more
tips regarding sample prep and and experiment
design.
Products
Affymetrix:
GeneChip Arrays
http://www.affymetrix.com/products/arrays/index.affx
Agilent Technologies:
Bioanalyzer
http://www.chem.agilent.com/Scripts/PDS.asp?lPage
=51
Oligo Array Kit
http://www.chem.agilent.com/Scripts/PDS.asp?lPage
=7307
Ambion:
RNAlater
http://www.ambion.com/techlib/resources/RNAlater/
index.html
Bio-Rad Laboratories:
Experion system
http://www.bio-rad.com/
BD Clontech:
Universal Total Reference RNA
http://www.clontech.com/clontech/products/literature
/pdf/brochures/UnivRNA.pdf
GE Healthcare Life Sciences:
Array products
http://www.amershambiosciences.com/
NanoDrop:
ND-1000 Spectrophotometer
http://www.nanodrop.com/products.html
NimbleGen Systems:
Array products
http://www.nimblegen.com/
14
Qiagen:
RNeasy Mini Spin Columns
http://www1.qiagen.com/Products/RnaStabilization
Purification/
TissueLyser
http://www1.qiagen.com/Products/Accessories/Tissue
Lyser/TissueLyser.aspx
Stratagene:
Reference RNA
http://www.stratagene.com/products/showCategory.
aspx?catId=193
Yasui Kikai:
Multi-Beads Shocker (cell disruptor)
http://www.yasuikikai.co.jp/company/e_index.html
Publications
Discovering Genomics, Proteomics,
and Bioinformatics
by A. Malcolm Campbell, Laurie J. Heyer
(September 2002) Benjamin Cummings;
ISBN: 0805347224
DNA Microarrays and Gene Expression
by Pierre Baldi, Wesley G. Hatfield
(October 2002) Cambridge University Press;
ISBN: 0521800226
Microarrays and Cancer Research
by Janet A. Warrington, Randy Todd, David Wong
(June 2002) Eaton Pub Co; ISBN: 1881299511
Microarray Quality Control
by Wei Zhang, Ilya Shmulevich, Jaako Astola
(April 2004) John Wiley & Sons; ISBN: 0471453447
Applying Genomic and Proteomic Microarray
Technology in Drug Discovery
by Robert S. Matson
(December 2003) CRC Press; ISBN: 0849314690
DNA Microarrays and Gene Expression
by Pierre Baldi, Wesley G. Hatfield
(October 2002) Cambridge University Press;
ISBN: 0521800226
Microarray Sample Prep Tech Guide
Genome Technology
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