Printing High Quality Microarrays

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Printing High Quality Microarrays

• Todd Martinsky
• todd_at_arrayit.com
• http//arrayit.com

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Outline

• Company background
• In the media
• Key personnel
• Established credibility
• Making the perfect microarray
• Its really a science, not an art
• Keys 1 through 5
• Conclusions

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• Privately held company
• Financially strong and profitable
• Fastest Growing Biotech in the America
• Microarray Technologies
• First company to create microarray products
• Superior technical expertise
• Recognized Leader in the field
• 10 year anniversary Nov. 1, 2003

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• Developed First Micro Spotting Pins for
  Microarray Manufacturing
• -Intellectual property from 1997
• Most Widely used Microarray Technology in the
  world Patent 6101946
• World leader in microarray consumables
• - Glass substrates, Pins, Hybridization
  cassettes, spotting solutions, sample prep kits,
  etc.

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Award Winning Service Quality
Silver Medal From DOD
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• Largest Circulationin the United States.
• 12 Companies mentioned
• 2 mentioned twice
• TeleChem Affymetrix

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Featured on NOVA
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How We Develop Products
Combine engineering principles with biological
expertise (Parallel Gene Analysis Methodology) to
systematically develop microarray products and
services.
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• Parallel Gene Analysis Methodology
• What you need to do and why, to complete the
  microarray lifecycle.
• Keep in simple, dont over-engineer
• Complicated systems and schemes break down and
  are typically expensive
• Critical thinking
• Customer Feedback
• End Result products, methods protocols
• Derived from our integrated In-House engineering
  and biology expertise
• Who are these people?

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In House Engineering Expertise

• Richard Martinsky Director of Engineering
• Designed Invented
• Hybridization Cassette
• Microarray Wash Station
• Stealth Chipmaker Micro Spotting Devices
• Process Engineering
• Substrate manufacturing
• Microarray Manufacturing
• Wash/Dry stations for Pins
• Microarray Scanners
• Microarray Robotics

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In House Microarray Expertise

• Mark Schena, Ph.D., Visiting Scholar
• Entire Microarray Industry based on first paper
• - Schena et al. Science. 270467. 1995.
• Editor of books on microarray Technology
• - (Oxford University Press, Biotechniques
  publishers)
• Wrote the textbook on microarrays
• - (Wiley Sons, Largest publisher of textbooks
  in the world)
• Recognized World Expert on Microarray Technology

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More Expertise

• Robin L. Stears, Ph.D.
• Senior Scientist/Director of Microarray
  Technology
• Utilized First commercially available microarryer
  Scanner -1997 (Laboratory of Dr. S. Gullans,
  Harvard Medical School)
• Developed Genisphere 3DNA microarray detection
  method (Stears et. Al, Physiological Genomics,
  2000)
• Head of Transcriptional Profiling Team,
  Affymetrix platform, Aventis Pharmaceuticals
  (2000-2001, Cambridge Genomics Center, MA)

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Thats me there

• Recognized leader with technological innovation,
  intellectual property
• Top names in the field-
• Mark Schena, Ph.D
• Robin L. Stears, Ph.D.
• Creating turn-key solutions for DNA Microarrays
  since 1996 (ArrayIt? Brand Products)

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Protocols on the web arrayit.com
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Making the Perfect Microarray
Our golden rule, If there is a variable in
your system, control it.
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This information is based on 8 years experience
providing technical support for microarray
manufacturing.
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Key Factors to Control

• Micro fluidic printing technology
• Robotics (including wash/dry station)
• Sample preparation
• Surface chemistry
• Environment
• If youve got a quality problem, I can guarantee
  its in 1 of these 5 areas.

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Printing High Quality Protein Microarrays

• 1. Printing Mechanisms

We should appreciate the fact that 1 picoliter is
to 1 liter as 1 cm is to 13 round-trips to the
moon!
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Our 12 Rules..being published later this year by
Kluwer
The Methodology of Printing Technologies
The principles that determine how spotting
technologies are used and interpreted. What
you need to do and why.
TeleChem/ArrayIt.com
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12 rules continued
1. Print uniform spots measured in
microns 2. Print individual spots in regular
array patterns that can be tracked by computer 3.
Easy to implement 4. Cost effective/affordable 5. 
Print without damaging the sample or surface
chemistry 6. Saturate the immobilization surface
chemistry at each spot location
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12 rules continued

• 7.  Amenable to high and low density
• 8.  Change spot sizes and sample volumes easily
• 9.  Load and deliver a specific amount of sample
  each time
• 10. Easy to fix and maintain, with no special
  tooling or tech visits required
• 11. Compatible with a variety of scientific
  applications
• 12. Print multiple samples, multiple times on
  multiple substrates with one low volume loading
  of sample

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Two Main Types
Consider the efficient use of sample when making
your choice! Contact Best for high numbers of
samples over many substrates Non-contact Low
numbers of samples over many, many substrates
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Non-Contact Types
Perkin Elmer others
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Non-Contact Types
To my knowledge, not used commercially Very high
heating of the sample makes it problematic Diffic
ult to change samples
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Non-Contact Types
Best for low numbers of samples and high numbers
of spots. Well made ceramic tips Different tips
for different spot sizes and volumes
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Non-Contact Types
Change delivery volume by firing multiple times
in the same spot location Spot size on par with
Pin spotting Typically slower than Pin spotting
since commercial systems are limited to 4-8
delivery nozzles Glass capillaries
Perkin Elmer Type
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Non Contact
IMITs TopSpot Uses an Piezo actuator and micro
fluidic channels. Industrial level manufacturing
of the same array
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Contact Printing Pin Ring
Advantages Multiple prints with 1
load Consistent and reliable Disadvantages Fixed
number of Pins (4) Large uptake volume Low
delivery vol. per spot Spring Loaded Pins No
flexibility to change spot size Complex actuators
not easy to fix (heat up during long runs) Large
source plate vol. 96 well plates No longer
supported by Affymetrix
What sticks to the tip of the pin as it passes
through the ring defines the amount of sample
delivered
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Split Pins and Quills
Advantages Multiple prints with 1 low volume of
load Patent owned by Incyte, but not
commercialized by them Flexible to change of
pins used only Can be replaced by user
Disadvantages Spring loaded (force on tips)
Tapping force to expel sample wears them out
quickly / variable deposition of sample Tip
tolerances uneven (ref., Brown patent) No
flexibility to change spot size Mistakes are
expensive
Split Pins (Many)
Tweezers / Quills (Schena et al.,1995)

• Variable sample uptake
• Forms a meniscus
• Tapping expels sample

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Patented Micro Spotting Pins by TeleChem
Advantages Multiple consistent prints with 1 low
vol. load Patented and commercialized by the same
organization with compatible consumables Flexible
to change of pins and spot size Easy to fix
Widely used Tight tolerances and quality
control Durable (under the right motion
parameters) Low volume of sample in source plate
(96 384 well)
Micro Spotting Pins

• Defined sample uptake (0.25, 0.6 or 1.25 ul)
• Sample at end of flat tip
• Substrate pulls off drop

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Stealth Micro Spotting Device Sub-nanoliter Vol.
Dispensing
PTO 6,101,946 Digitally controlled manufacturing
355X
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Spotting Sequence
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2 Micron Tolerance
Mechanically identical parts perform identical
tasks
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Typical Results
Spot 1
Spot 200
Cy3 Labeled oligo in Micro Spotting
Solution-1 equal spot sizes, equal signal
intensities
ArrayIt? Stealth 3 Pin
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Analysis of Typical Results
NoteWhen the key elements 1-5 are controlled
properly
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Printing High Quality Protein Microarrays

• 2. Sample Preparation

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Contaminates in spotted sample

• Prohibit samples from immobilizing on the
  microarray printing services
• Prohibit interaction between array elements and
  probes
• Cause background noise
• Can clog pins and other printing mechanisms
• Ruin spot morphology

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PCR PurificationMembrane vs. ETOH Precipitation
Data
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PCR and Fluorescent Probe Purification
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Spotting Buffer
Qualities of a good spotting buffer

• Print even, small, round spots
• Disperses the sample evenly within the spot
• Promote sample binding to the array surface
• Retard evaporation within the source plates
• Dry evenly, perhaps not dry at all
• Wash away easily
• Optimize attachment
• Dry down and re-suspend
• Visual after spotting regardless of surface
• Stabilize sample for long term storage

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Microplates and Samples

• 384 round wells, not 96 wells
• better for avoiding evaporation
• Rigid polypropylene construction
• V or U bottom shaped wells
• 3-15 microliters of sample per well

U U
Sample
Polypropylene
Polystyrene
Does not bind DNA
Binds DNA
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Making the Perfect Microarray

• 3. Robotics

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A good microarrayer has

• Accuracy and repeatability on the micron level
• Computer controlled GUI for easy programming and
  sample tracking
• Good wash/dry station between sample changes to
  eliminate cross contamination between samples
• Humidity and temperature control in a closed
  cleanroom level positive pressure environment

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A good example
Self contained environmental (humidity)
controlled chamber to clean room level quality
dynamicdevices Oasis
TeleChem/ArrayIt.com
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Avoiding Sample Carryover
Use multiple wash/dry cycles, never dry the
printing mechanism until the last wash cycle is
complete!
Is the job of the wash/dry station on the
microarrayer
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Minimum Software Requirements

• Number of sample delivery mechanisms and the
  center-to-center spacing of said mechanisms
  (4.5mm or 9mm centers).
• The total number of samples to be printed
• Offsets relative to the substrate
• Number of replicates of each sample
• Center-to-center distance between spots
• Number of columns and rows
• Number of substrates/slides to be printed
• Wash/dry parameters for the printing mechanisms
  between printing cycles.
• Mapping!!!!!

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Easy Programming Example
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Personal Microarray System
1000 samples every 2 hours over 14 substrates
may be high enough throughput?
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Making the Perfect Microarray

• 4. Sample Immobilization

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3D (absorption) vs. 2D (covalent) Surfacesin
general

• Advantages of 3D (membranes, filters gels)
• High binding capacity (absorption)
• Compatible with fluorescent, chemiluminescent,
  colorimetric, radioactive detection
• Longer history of use (comfort level for users)
• Less expensive labeling reagents and reading
  equipment (colorimetric)

14µm thick nitrocellulose-based coating. Electron
micrograph image above, the uniform pore
structure provides a large, 3-dimensional surface
area for protein binding. The 3-dimensional
surface quantitatively binds arrayed
proteins www.schleicher-schuell.com
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3D (absorption) vs. 2D (covalent) Surfacesin
general

• Advantages of 2D
• Better defined spot morphology(no diffusion)
• Inherent lower background fluorescence (glass)
• High specificity
• Non-porous surface (no place to trap any
  contaminate in processing)
• Covalent and/or specific binding for more
  stringent processing conditions

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• Homogenous distribution of capture reactive
  groups across the entire surface is critical for
  attaching the same amount of sample at each array
  location
• The printing mechanism must saturate capture
  groups at each spot location, since what does not
  bind washes away in processing

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Effects of Spotting Surface Morphology
Homogenous
Heterogeneous
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2D, Amino Silane
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2D, Aldehyde coupling
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2D, Epoxy Coupling
Reacts with Primary amines, but does not require
dehydration
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Making the Perfect Microarray

• 5. Environment

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Environmental Keys

• Cleanliness
• Temperature
• Humidity
• Clean rooms helpbut arent necessary

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Easy Ways Clean Up

• Hepa Air Filters from your local hardware store
• Replace old ceiling tiles
• Keep arrayers away from air vents or add filters
  to incoming air
• Buy an arrayer with good environmental control
• Have a dedicated microarraying environment

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Note on Humidity

• Take it out of the room (work comfort) and add it
  to the arraying chamber (sample evaporation)
• It is too difficult to control an entire room,
  but easy inside a small arraying chamber

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Notes on Gloves

• Avoid latex gloves that leave contaminating
  protein residue
• Use powder free gloves only
• Synthetic rubber

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Class-100 Clean Room Environment

• Dust-Free
• - Air filtered by ULPA Filtration
• Precisely Controlled
• - Humidity
• Temperature

TeleChem/ArrayIt.com
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When the key elements are controlled
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It works Microarray Resource Center http//array
it.com/e-library/ 1,965 total publications for
"microarray"
Miragene Inc.unpublished
MacBeath Schrieber, Science, 2891760, 2000
Schleicher-Schuellunpublished
Stears, et al. Nature Med 2003.
Zhu, H. et al. (2001)
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Personal Microarray Core Facility - 30K instead
of 150K
In development a complete line of hardware,
software, and consumables (colorimetric, single
color, enzymatic labeling)
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MicroarraysUniversal Biochemistry Platforms
Proteins
DNA
Peptides
Lipids
Small molecules
Carbohydrates