Printing High Quality Microarrays
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Transcript of Printing High Quality Microarrays
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Printing High Quality Printing High Quality MicroarraysMicroarrays
Todd [email protected]://arrayit.com
arrayit.coarrayit.comm
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Outline Outline Company backgroundIn the mediaKey personnel
– Established credibilityMaking the perfect microarray
– It’s really a science, not an art– Keys 1 through 5
Conclusions
arrayit.coarrayit.comm
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• Privately held company Privately held company
• Financially strong and profitableFinancially strong and profitable
• Fastest Growing Biotech in the AmericaFastest Growing Biotech in the America
• Microarray TechnologiesMicroarray Technologies
• First company to create microarray productsFirst company to create microarray products
• Superior technical expertiseSuperior technical expertise
• Recognized Leader in the fieldRecognized Leader in the field
•10 year anniversary Nov. 1, 200310 year anniversary Nov. 1, 2003
arrayit.coarrayit.comm
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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 & QualityAward Winning Service & Quality
Silver Medal
From
DOD
arrayit.coarrayit.comm
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Largest Circulationin the United States.
12 Companies mentioned
2 mentioned twice TeleChem &
Affymetrix
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Featured on NOVAFeatured on NOVA
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How We Develop ProductsHow We Develop Products
Combine engineering principles with Combine engineering principles with biological expertise (Parallel Gene biological expertise (Parallel Gene Analysis Methodology) to systematically Analysis Methodology) to systematically develop microarray products and develop microarray products and services.services.
TeleChem / arrayit.comarrayit.com
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Parallel Gene Analysis Methodology– What you need to do and why, to complete
the microarray lifecycle. – Keep in simple, don’t 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 ExpertiseIn 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 ExpertiseIn House Microarray Expertise
• Mark Schena, Ph.D., Visiting Scholar
• Entire Microarray Industry based on first paper:
- Schena et al. Science. 270:467. 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…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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• 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)
That’s me there
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Protocols on the web – Protocols on the web – arrayit.comarrayit.com
Summary by Month
MonthDaily Avg Monthly Totals
Hits Files Pages Visits Sites KBytes Visits Pages Files Hits
Jan 2003 32472 24052 4290 648 3741 2356350 6485 42907 240527 324726
Dec 2002 29811 20335 4644 594 8890 6346647 18414 143979 630386 924152
Nov 2002 23953 16355 3427 411 7142 5439463 12338 102820 490659 718613
Oct 2002 27323 18979 4415 670 9638 7636378 20776 136874 588362 847016
Sep 2002 24863 16711 4399 643 8107 6593075 19300 131996 501352 745900
Aug 2002 9086 6068 1515 269 3766 2356590 8368 46976 188134 281672
Jul 2002 21104 13970 2917 531 7157 5451430 16483 90457 433090 654224
Jun 2002 23863 16185 4306 663 8284 5995133 19893 129206 485569 715902
May 2002 26011 16551 4066 688 8227 6258849 21331 126054 513103 806367
Apr 2002 26316 17642 3565 624 8087 6204948 18738 106965 529267 789480
Mar 2002 23505 15509 3302 566 7917 5449506 17574 102367 480808 728656
Feb 2002 24843 16755 3350 549 7243 5398343 15399 93814 469161 695630
Totals6548671
219509
9125441
5555041
8823233
8
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Making the Perfect Making the Perfect MicroarrayMicroarray
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 ControlKey Factors to Control
1. Micro fluidic printing technology
2. Robotics (including wash/dry station)
3. Sample preparation
4. Surface chemistry
5. Environment
• If you’ve got a quality problem, I can guarantee it’s in 1 of these 5 areas.
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1. Printing Mechanisms
Printing High QualityPrinting High Quality Protein Microarrays Protein Microarrays
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 Our 12 Rules…..being published later this year by Kluwerby Kluwer
The principles that determine how spotting technologies are used and interpreted.
What you need to do and why.
TeleChem/ArrayIt.comArrayIt.com
The Methodology of Printing Technologies
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12 rules continued…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…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 TypesTwo Main TypesConsider 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 TypesNon-Contact Types
Perkin Elmer & others
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Non-Contact TypesNon-Contact Types
To my knowledge, not used commercially
Very high heating of the sample makes it problematic
Difficult to change samples
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Non-Contact TypesNon-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 TypesNon-Contact TypesChange 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 ContactNon Contact
IMIT’s TopSpot Uses an Piezo actuator and micro fluidic channels.
Industrial level manufacturing of the same array
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Contact Printing – Pin & RingContact 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 PinsSplit Pins and Quills and QuillsTweezers / QuillsTweezers / Quills
(Schena et al.,1995)(Schena et al.,1995)Split PinsSplit Pins
(Many)(Many)
•Variable sample uptake
•Forms a meniscus
•Tapping expels sample
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
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Patented Patented Micro Spotting Pins by TeleChemMicro Spotting Pins by TeleChem
Micro Spotting PinsMicro Spotting Pins
•Defined sample uptake (0.25, 0.6 or 1.25 ul)
•Sample at end of flat tip
•Substrate pulls off drop
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)
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Stealth Micro Spotting Device Stealth Micro Spotting Device Sub-nanoliter Vol. DispensingSub-nanoliter Vol. Dispensing
355XPTO# 6,101,946Digitally controlled manufacturing
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Spotting Sequence…Spotting Sequence…
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++ 2 Micron Tolerance
Mechanically identical parts Mechanically identical parts perform identical tasksperform identical tasks
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Typical ResultsTypical Results
Spot # 200
Cy3 Labeled oligo in Micro Spotting Solution-1equal spot sizes, equal signal intensities
ArrayItArrayIt Stealth 3 Stealth 3 PinPin
Spot #1
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Analysis of Typical ResultsAnalysis of Typical Results
QuantArray analysis software (Packard Biosystems) data for 300 spots.
Diameter Circularity Uniformity
Average 113.2 0.95 1.00
STD 4.2 0.01 0.00
CV 0.04 0.01 0.00
Note:When the key elements 1-5 are controlled properly
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2. Sample Preparation
Printing High QualityPrinting High Quality Protein Microarrays Protein Microarrays
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Contaminates in Contaminates in spotted sample…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 PurificationPCR PurificationMembrane vs. ETOH Membrane vs. ETOH
Precipitation DataPrecipitation Data
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PCR and Fluorescent Probe PCR and Fluorescent Probe PurificationPurification
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Spotting BufferSpotting 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
Qualities of a good spotting buffer:Qualities of a good spotting buffer:
BadBad Better Better
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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 UPolypropylene Polystyrene
Sample
Does not bind DNA Binds DNA
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Making the Perfect Making the Perfect MicroarrayMicroarray
3. Robotics
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A good microarrayer has…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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Self contained environmental (humidity) controlled chamber to clean room level quality
TeleChem/ArrayIt.comArrayIt.comdynamicdevices Oasis
A good example…A good example…
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Avoiding Sample CarryoverAvoiding Sample Carryover
Is the job of the wash/dry station on the microarrayer
Use multiple wash/dry cycles, never dry the printing mechanism until the last wash cycle is complete!
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Minimum Software Minimum Software RequirementsRequirements
•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 ExampleEasy Programming Example
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Personal Microarray Personal Microarray SystemSystem
~1000 samples every 2 hours over 14 substrates may be high enough throughput?
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Making the Perfect MicroarrayMaking the Perfect Microarray
4. Sample Immobilization
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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 proteinswww.schleicher-schuell.com
3D (absorption) vs. 2D (covalent) Surfaces3D (absorption) vs. 2D (covalent) Surfacesin general…in general…
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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
Angstroms
Inte
nsit
y S
cale
50.0
25.0
0.0
3D (absorption) vs. 2D (covalent) Surfaces3D (absorption) vs. 2D (covalent) Surfacesin general…in general…
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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 Effects of Spotting Surface & MorphologySurface & Morphology
HomogenousHomogenous HeterogeneousHeterogeneous
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2D, Amino Silane
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2D, Aldehyde coupling2D, Aldehyde coupling
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2D, Epoxy Coupling2D, Epoxy Coupling
Reacts with Primary amines, but does not require dehydration
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Making the Perfect Making the Perfect MicroarrayMicroarray
5. Environment
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Environmental Keys…Environmental Keys…
• Cleanliness• Temperature • Humidity• Clean rooms help
but aren’t necessary
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Easy Ways “Clean Up”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 HumidityNote 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 GlovesNotes 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 Class-100 Clean Room EnvironmentEnvironment
Dust-Free Dust-Free
- Air filtered by - Air filtered by ULPA FiltrationULPA Filtration
Precisely Precisely ControlledControlled
- Humidity- Humidity
-Temperature Temperature ––
TeleChem/ArrayIt.comArrayIt.com
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When the key elements When the key elements are controlled…are controlled…
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It works…
Microarray Resource Center™
http://arrayit.com/e-library/
1,965 total publications for "microarray"
Haab, et al, Genome Biology 2001 2(2): research 0004.1-0004.13
MacBeath & Schrieber, Science, 289:1760, 2000
Zhu, H. et al. (2001)
Stears, et al. Nature Med 2003.
Schleicher-Schuellunpublished
Miragene Inc.unpublished
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Personal Microarray Core Facility Personal Microarray Core Facility - $30K instead of $150K- $30K instead of $150K
In development a complete
line of hardware,
software, and consumables
(colorimetric, single color, enzymatic labeling)
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Microarrays:Microarrays:Universal Biochemistry PlatformsUniversal Biochemistry Platforms
PeptidesPeptides ProteinsProteins
Carbohydrates
LipidsLipids
Small moleculesSmall molecules
DNADNA