Microarray Technology

- a brief introduction -

Markus Panhuysen

MPI of Psychiatry
GSF-Research Center for Environment and Health

g
Molecular Neurogenetics Group
Institute of Developmental Genetics
Kraepelinstr. 2
Ingolstädter Landstraße 1
80804 Munich
85764 Neuherberg
 Microarray Technology is a powerful tool
to monitor gene expression or gene expression
changes of hundreds or thousands of genes
in a single experiment.

MPI of Psychiatry
GSF-Research Center for Environment and Health

g
Molecular Neurogenetics Group
Institute of Developmental Genetics
Kraepelinstr. 2
Ingolstädter Landstraße 1
80804 Munich
85764 Neuherberg
 Microarrays

-> hundreds or thousands of gene specific probes (cDNA or Oligo-

nucleotides) fixed on a solid support (usually glass or plastic slides
or membranes)
-> self-made or commercially available (Agilent, Codelink, Clonetech,
Stratagene, etc. ; or GeneChips from Affymetrix)

MPI of Psychiatry
GSF-Research Center for Environment and Health

g
Molecular Neurogenetics Group
Institute of Developmental Genetics
Kraepelinstr. 2
Ingolstädter Landstraße 1
80804 Munich
85764 Neuherberg
 Applications of Microarrays...

* Expression profiling

* Detection of changes in gene expression

(simple comparison or multiple conditions, time course experiments,...)

* Diagnostic tool
(tumor classification, genomic Microarrays: detection of chromosomal
rearragements, deletions or duplications)

* Other applications
(SNP analysis, detection of methylation patterns,...)

MPI of Psychiatry
GSF-Research Center for Environment and Health

g
Molecular Neurogenetics Group
Institute of Developmental Genetics
Kraepelinstr. 2
Ingolstädter Landstraße 1
80804 Munich
85764 Neuherberg
 Term definitions...

PROBE: cDNA or oligonocleotide attached to the array surface

TARGET: cDNA or aRNA within the hybridization mix (which

can hybridize to the complementary probe strand)

MPI of Psychiatry
GSF-Research Center for Environment and Health

g
Molecular Neurogenetics Group
Institute of Developmental Genetics
Kraepelinstr. 2
Ingolstädter Landstraße 1
80804 Munich
85764 Neuherberg
 Experiment Overview
Array Production Sample Production
cells / tissues
RNA
(amplification)
#1
Labeling
#2

Hybridization
Stringency Washes

Scanning
Quantification

Normalization
Analysis
Annotation
MPI of Psychiatry
GSF-Research Center for Environment and Health

g
Molecular Neurogenetics Group
Institute of Developmental Genetics
Kraepelinstr. 2
Ingolstädter Landstraße 1
80804 Munich
85764 Neuherberg
 Experiment Overview
Array Production Sample Production
cells / tissues
RNA
(amplification)
#1
Labeling
#2

Hybridization
Stringency Washes

Scanning
Quantification

Normalization
Analysis
Annotation
MPI of Psychiatry
GSF-Research Center for Environment and Health

g
Molecular Neurogenetics Group
Institute of Developmental Genetics
Kraepelinstr. 2
Ingolstädter Landstraße 1
80804 Munich
85764 Neuherberg
 Production of cDNA Microarrays
Bacterial Clone Library Spotting / Printing

cDNA Amplification (PCR) Probe Crosslinking

PCR Product Purification/

transfer in Spotting Buffer Blocking /
Prehybridization
Rearraying
(96well  384well)

MPI of Psychiatry
GSF-Research Center for Environment and Health

g
Molecular Neurogenetics Group
Institute of Developmental Genetics
Kraepelinstr. 2
Ingolstädter Landstraße 1
80804 Munich
85764 Neuherberg
High Throughput System for cDNA Amplification

MPI of Psychiatry
GSF-Research Center for Environment and Health

g
Molecular Neurogenetics Group
Institute of Developmental Genetics
Kraepelinstr. 2
Ingolstädter Landstraße 1
80804 Munich
85764 Neuherberg
 The Spotter

courtesy of TeleChem

MPI of Psychiatry
GSF-Research Center for Environment and Health

g
Molecular Neurogenetics Group
Institute of Developmental Genetics
Kraepelinstr. 2
Ingolstädter Landstraße 1
80804 Munich
85764 Neuherberg
 Experiment Overview
Array Production Sample Production
cells / tissues
RNA
(amplification)
#1
Labeling
#2

Hybridization
Stringency Washes

Scanning
Quantification

Normalization
Analysis
Annotation
MPI of Psychiatry
GSF-Research Center for Environment and Health

g
Molecular Neurogenetics Group
Institute of Developmental Genetics
Kraepelinstr. 2
Ingolstädter Landstraße 1
80804 Munich
85764 Neuherberg
 RNA
isolated from cells or tissues of interest
e.g., from RA treated cells and control cells

RNA Quality Check:

by denaturing gel
electrophoresis

8 hrs retinoic acid 8 hrs control

treatment treatment
or RNA LabChip
RNA extraction

RNARA RNACtrl

courtesy of Agilent

MPI of Psychiatry
GSF-Research Center for Environment and Health

g
Molecular Neurogenetics Group
Institute of Developmental Genetics
Kraepelinstr. 2
Ingolstädter Landstraße 1
80804 Munich
85764 Neuherberg
 RNA Labeling
usually involves a reverse transcription step
optional: RNA amplification

 direct or indirect incorporation of fluorescent dyes, radioactivity or

epitope tags (e.g, biotin); single color or dual color experiments

RNARA RNACtrl
AAAAAA AAAAAA

TTTTTT TTTTTTT labeled cDNAs

MPI of Psychiatry
GSF-Research Center for Environment and Health

g
Molecular Neurogenetics Group
Institute of Developmental Genetics
Kraepelinstr. 2
Ingolstädter Landstraße 1
80804 Munich
85764 Neuherberg
 Hybridization
incubation of Microarrays for 16 hrs or more with a hybe-mix
containing the labeled cDNAs

TTTTTT
TTTTTTT
TTTTTTT TTTTTT
TTTTTTT
TTTTTT
Mix of labeled cDNAs (aRNAs)

Microarray

MPI of Psychiatry
GSF-Research Center for Environment and Health

g
Molecular Neurogenetics Group
Institute of Developmental Genetics
Kraepelinstr. 2
Ingolstädter Landstraße 1
80804 Munich
85764 Neuherberg
 Experiment Overview
Array Production Sample Production
cells / tissues
RNA
(amplification)
#1
Labeling
#2

Hybridization
Stringency Washes

Scanning
Quantification

Normalization
Analysis
Annotation
MPI of Psychiatry
GSF-Research Center for Environment and Health

g
Molecular Neurogenetics Group
Institute of Developmental Genetics
Kraepelinstr. 2
Ingolstädter Landstraße 1
80804 Munich
85764 Neuherberg
 Stringency Washes, Drying, Scanning and
Quantification

MPI of Psychiatry
GSF-Research Center for Environment and Health

g
Molecular Neurogenetics Group
Institute of Developmental Genetics
Kraepelinstr. 2
Ingolstädter Landstraße 1
80804 Munich
85764 Neuherberg
 Log-Plot
Log2(ICy3)

Log2(ICy5)

MPI of Psychiatry
GSF-Research Center for Environment and Health

g
Molecular Neurogenetics Group
Institute of Developmental Genetics
Kraepelinstr. 2
Ingolstädter Landstraße 1
80804 Munich
85764 Neuherberg
 Data Analysis

MPI of Psychiatry
GSF-Research Center for Environment and Health

g
Molecular Neurogenetics Group
Institute of Developmental Genetics
Kraepelinstr. 2
Ingolstädter Landstraße 1
80804 Munich
85764 Neuherberg
 Experiment Design
* single color/labeling or dual color experiment

* direct sample labeling or indirect sample labeling

* with or without Dye Swap

* sample by sample or sample pools

* technical replicates or biological replicates

* with or without reference RNA

* in case of multiple conditions: Which samples should be

directly compared?

MPI of Psychiatry
GSF-Research Center for Environment and Health

g
Molecular Neurogenetics Group
Institute of Developmental Genetics
Kraepelinstr. 2
Ingolstädter Landstraße 1
80804 Munich
85764 Neuherberg
 Sample complexity affects the efficiency of
microarray experiments...

Gene expression changes in a subset of cells within a sample are

diluted by unaltered expression in other cells, resulting in reduced
measurable expression changes.

From the technical point of view, samples should be as little

complex as possible.

MPI of Psychiatry
GSF-Research Center for Environment and Health

g
Molecular Neurogenetics Group
Institute of Developmental Genetics
Kraepelinstr. 2
Ingolstädter Landstraße 1
80804 Munich
85764 Neuherberg
 Differential expression in a cell line...

Wurmbach et al., 2002

MPI of Psychiatry
GSF-Research Center for Environment and Health

g
Molecular Neurogenetics Group
Institute of Developmental Genetics
Kraepelinstr. 2
Ingolstädter Landstraße 1
80804 Munich
85764 Neuherberg
 ... in the hypothalamus...

Wurmbach et al., 2002

MPI of Psychiatry
GSF-Research Center for Environment and Health

g
Molecular Neurogenetics Group
Institute of Developmental Genetics
Kraepelinstr. 2
Ingolstädter Landstraße 1
80804 Munich
85764 Neuherberg
 ...and in the cerebral cortex...

Wurmbach et al., 2002

MPI of Psychiatry
GSF-Research Center for Environment and Health

g
Molecular Neurogenetics Group
Institute of Developmental Genetics
Kraepelinstr. 2
Ingolstädter Landstraße 1
80804 Munich
85764 Neuherberg
 Sources of Variation within
Microarray Experiments...

biological technical
animal to animal differences caused by the experimentator,
in gene expression by equipment measuring errors,
by technical limitations,
by bad protocols,
...
systematic unsystematic,
"noise"

MPI of Psychiatry
GSF-Research Center for Environment and Health

g
Molecular Neurogenetics Group
Institute of Developmental Genetics
Kraepelinstr. 2
Ingolstädter Landstraße 1
80804 Munich
85764 Neuherberg
 Sources of errors / technical variation
Array production: Clone quality

...clone sequence incorrect

...clone contaminated (with other clones)

Nucleic Acids Research, 2001, Vol. 29, No. 2 582-588

Assessment of clone identity and sequence fidelity
for 1189 IMAGE cDNA clones
Robe rt G. Halgren , Mark R. Fie lde n, Cora J. Fong and Timothy R. Zachare wski
Departm ent of Biochemistry and Molecular Biology , National Food Safety and Toxicology Center, and Institute for Environmental Toxicology , Michigan State University , East Lansing, MI 48824-1319,
USA

...After isolation of plasmid DNA from 1189 bacterial stock cultures,

only 62.2% were uncontaminated and contained cDNA inserts that
had significant sequence identity to published data for the ordered
clones....
MPI of Psychiatry
GSF-Research Center for Environment and Health

g
Molecular Neurogenetics Group
Institute of Developmental Genetics
Kraepelinstr. 2
Ingolstädter Landstraße 1
80804 Munich
85764 Neuherberg
 Sources of errors / technical variation
Array production: cDNA probe amplification
...does not work for some clones
...results in too low concentration of the PCR product

MPI of Psychiatry
GSF-Research Center for Environment and Health

g
Molecular Neurogenetics Group
Institute of Developmental Genetics
Kraepelinstr. 2
Ingolstädter Landstraße 1
80804 Munich
85764 Neuherberg
 Sources of errors / technical variation
Array production: Spotting process
pin printing failures
spot size varies too much, probes mix on the slides
bad spot morphology, tadpole effects, horseshoes etc.
pin-specific artefacts
Array production: Slides
scratches, dust or other contaminations
inhomogenous surface coating
surface chemistry and spotting buffer

Array production: Blocking / Prehybridization

array surface not completely inactivated
(results in unspecific probe binding)

MPI of Psychiatry
GSF-Research Center for Environment and Health

g
Molecular Neurogenetics Group
Institute of Developmental Genetics
Kraepelinstr. 2
Ingolstädter Landstraße 1
80804 Munich
85764 Neuherberg
 some examples...

the good... ...the bad... ...the ugly

MPI of Psychiatry
GSF-Research Center for Environment and Health

g
Molecular Neurogenetics Group
Institute of Developmental Genetics
Kraepelinstr. 2
Ingolstädter Landstraße 1
80804 Munich
85764 Neuherberg
 MPI of Psychiatry
GSF-Research Center for Environment and Health

g
Molecular Neurogenetics Group
Institute of Developmental Genetics
Kraepelinstr. 2
Ingolstädter Landstraße 1
80804 Munich
85764 Neuherberg
 courtesy of Jean Yang

MPI of Psychiatry
GSF-Research Center for Environment and Health

g
Molecular Neurogenetics Group
Institute of Developmental Genetics
Kraepelinstr. 2
Ingolstädter Landstraße 1
80804 Munich
85764 Neuherberg
 courtesy of Jean Yang

MPI of Psychiatry
GSF-Research Center for Environment and Health

g
Molecular Neurogenetics Group
Institute of Developmental Genetics
Kraepelinstr. 2
Ingolstädter Landstraße 1
80804 Munich
85764 Neuherberg
 Sources of errors / technical variation
RNA
RNA degradation
Sloppy sample isolation
(spatial variation of tissue punches from sections, variation
in sample quality due to contaminations)
Labeling Efficiency too low or varying among samples
Labeling Bias (e.g., due to direct incorporation of CyeDyes)
Inhomogenous hybridization / washing conditions
Spatial bias caused by the Scanner
Quantification method not appropriate for specific spot
characteristics
Inappropriate normalization methods (e.g, subtraction of nonadditive
backgrounds)
MPI of Psychiatry
GSF-Research Center for Environment and Health

g
Molecular Neurogenetics Group
Institute of Developmental Genetics
Kraepelinstr. 2
Ingolstädter Landstraße 1
80804 Munich
85764 Neuherberg
 Quantification methods

Fixed circle

Adaptive Circle

Adaptive Shape
(Edge detection/ Seeded
Region Growing)

Histogram
(Adaptive threshold based)

courtesy of Jean Yang

MPI of Psychiatry
GSF-Research Center for Environment and Health

g
Molecular Neurogenetics Group
Institute of Developmental Genetics
Kraepelinstr. 2
Ingolstädter Landstraße 1
80804 Munich
85764 Neuherberg
 The Quantification method does matter...
Spot.nbg Spot.morph

Spot.valley ScanAlyze

courtesy of Jean Yang

MPI of Psychiatry
GSF-Research Center for Environment and Health

g
Molecular Neurogenetics Group
Institute of Developmental Genetics
Kraepelinstr. 2
Ingolstädter Landstraße 1
80804 Munich
85764 Neuherberg
 MPI of Psychiatry
GSF-Research Center for Environment and Health

g
Molecular Neurogenetics Group
Institute of Developmental Genetics
Kraepelinstr. 2
Ingolstädter Landstraße 1
80804 Munich
85764 Neuherberg
 Thanks!

MPI of Psychiatry
GSF-Research Center for Environment and Health

g
Molecular Neurogenetics Group
Institute of Developmental Genetics
Kraepelinstr. 2
Ingolstädter Landstraße 1
80804 Munich
85764 Neuherberg
 Commercial Microarray Solutions...
... are standardized (array production and experimental protocols)

... are quality controlled

... usually provide tools for quantification, normalization, analysis,

visualization, data warehousing, project management

... are expensive

... are either inflexible (only standard sets are available)

... or even more expensive (custom arrays)

MPI of Psychiatry
GSF-Research Center for Environment and Health

g
Molecular Neurogenetics Group
Institute of Developmental Genetics
Kraepelinstr. 2
Ingolstädter Landstraße 1
80804 Munich
85764 Neuherberg
 In-House Microarray Solutions...
... are cheaper than commercial solutions
 if big amounts of experiments are to be performed

... provide maximum control of any parameter involved

... are very flexible

 protocols can be adapted to individual demands,e.g, performing experiments
using specific subsets of probes
... enable the use of special applications
such as suppressive subtractive library experiments, RNA Arrays, etc
.
... have to be established (protocols, analysis tools, ...)
There are as many different experimental protocols
as there are different Microarray Laboratories
MPI of Psychiatry
GSF-Research Center for Environment and Health

g
Molecular Neurogenetics Group
Institute of Developmental Genetics
Kraepelinstr. 2
Ingolstädter Landstraße 1
80804 Munich
85764 Neuherberg