Printing High Quality Protein Microarrays
Transcript of Printing High Quality Protein Microarrays
Printing High Quality Printing High Quality MicroarraysMicroarrays
Todd [email protected]://arrayit.com
arrayit.comarrayit.com
Making the Perfect Making the Perfect MicroarrayMicroarray
Our golden rule,
“If there is a variable in your system,control it.”
This information is based on 8 years experience providing technical support for microarray manufacturing….
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.
Printing High QualityPrinting High QualityProtein MicroarraysProtein 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!
The Methodology of PrintingTechnologies
The principles that determine how spotting technologies are used and interpreted.
What you need to do and why.
Our 12 Rules…..being published later this year Our 12 Rules…..being published later this year by Kluwerby Kluwer
TeleChem/ArrayIt.comArrayIt.com
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
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
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
NonNon--Contact TypesContact Types
To my knowledge, not used commercially
Very high heating of the sample makes it problematic
Difficult to change samples
NonNon--Contact TypesContact Types
Best for low numbers of samples and high numbers of spots.
Well made ceramic tips
Different tips for different spot sizes and volumes
NonNon--Contact TypesContact 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
Non ContactNon Contact
IMIT’s TopSpot Uses an Piezo actuator and micro fluidic channels.
Industrial level manufacturing of the same array
Contact Printing Contact Printing –– Pin & RingPin & 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
Split PinsSplit Pinsand Quillsand Quills
Tweezers / 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
Patented Patented Micro Spotting Pins by TeleChemMicro 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 PinsMicro Spotting Pins
•Defined sample uptake (0.25, 0.6 or 1.25 ul)•Sample at end of flat tip•Substrate pulls off drop
Stealth Micro Spotting Device Stealth Micro Spotting Device SubSub--nanoliter Vol. Dispensingnanoliter Vol. Dispensing
PTO# 6,101,946Digitally controlled manufacturing 355X
++ 2 Micron Tolerance
Mechanically identical parts Mechanically identical parts perform identical tasksperform identical tasks
Typical ResultsTypical Results
Spot #1
Spot # 200
Cy3 Labeled oligo in Micro Spotting Solution-1equal spot sizes, equal signal intensities
ArrayItArrayIt Stealth 3 PinStealth 3 Pin
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
Printing High QualityPrinting High QualityProtein MicroarraysProtein Microarrays
2. Sample Preparation
Contaminates spotted Contaminates spotted sample…sample…
Prohibit samples from immobilizing on the microarray printing servicesProhibit interaction between array elements and probesCause background noiseCan clog pins and other printing mechanisms
Ruin spot morphology
PCR PurificationPCR PurificationMembrane vs. ETOH Membrane vs. ETOH
Precipitation DataPrecipitation Data
– 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 BetterBetter
Spotting BufferSpotting Buffer
Microplates and Samples384 round wells, not 96 wells
better for avoiding evaporationRigid polypropylene constructionV or U bottom shaped wells3-15 microliters of sample per well
Polypropylene Polystyrene
Sample
Does not bind DNA Binds DNA
A good microarrayer has…A good microarrayer has…
Accuracy and repeatability on the micron levelComputer controlled GUI for easy programming and sample trackingGood wash/dry station between sample changes to eliminate cross contamination between samplesHumidity and temperature control in a closed “cleanroom” level positive pressure environment
A good example…A good example…
ChipWriter Pro from Bio-Rad
Self contained environmental (humidity) controlled chamber to clean room level quality
TeleChem/ArrayIt.comArrayIt.com
Avoiding Sample CarryoverAvoiding Sample CarryoverUse 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
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!!!!!
Personal Microarray Personal Microarray SystemSystem
~1000 samples every 2 hours over 14 substrates may be high enough throughput?
3D (absorption) vs. 2D (covalent) Surfaces3D (absorption) vs. 2D (covalent) Surfacesin general…in 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 proteinswww.schleicher-schuell.com
3D (absorption) vs. 2D (covalent) Surfaces3D (absorption) vs. 2D (covalent) Surfacesin general…in general…
Angstroms
Inte
ns i
t y S
cale 50.0
25.0
0.0
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
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
Effects of Spotting Effects of Spotting Surface & MorphologySurface & Morphology
HomogenousHomogenous HeterogeneousHeterogeneous
Environmental Keys…Environmental Keys…
• Cleanliness• Temperature • Humidity• Clean rooms help
but aren’t necessary
Easy Ways “Clean Up”Easy Ways “Clean Up”
Hepa Air Filters from your local hardware storeReplace old ceiling tilesKeep arrayers away from air vents or add filters to incoming airBuy an arrayer with good environmental controlHave a dedicated microarraying environment
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
Notes on GlovesNotes on Gloves
Avoid latex gloves that leave contaminating protein residue
Use powder free gloves only
Synthetic rubber
ClassClass--100 Clean Room 100 Clean Room EnvironmentEnvironment
DustDust--Free Free
-- Air filtered by Air filtered by ULPA FiltrationULPA Filtration
Precisely ControlledPrecisely Controlled-- HumidityHumidity--Temperature Temperature ––
TeleChem/ArrayIt.comArrayIt.com
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
Miragene Inc.unpublished
MacBeath & Schrieber, Science, 289:1760, 2000
Schleicher-Schuellunpublished
Stears, et al. Nature Med 2003.
Zhu, H. et al. (2001)
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)