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Overview
Microarrays on chemically treated glass slides are made by dispensing
a small amount of oligonucleotide or PCR product using a solid dispensing
mechanism. Common dispensing mechanisms are tweezers, rings, split
pins, inkjets, or microspotting pins. In the most common system,
using microspotting pins with small chambers, a small volume (typically
between 100 and 300nl) is taken up from 384-well PCR plates (“spotting
plates”) containing less than 10ml per well. There may be one or
more of these pins taking up DNA simultaneously; up to 32 pins are
possible on many arrayers. When a pin touches the surface of a
slide treated with a special coating, a small volume of DNA is ‘printed’
onto the slide (about 1/3 to 1/2nl). The pins are mounted in a
gridded head, 4.5mm apart, at the end of a robotic arm, which in
turn is controlled by a software program directing the arm to create
a specified grid of spots.
Metagrids
and subgrids
Because of the configuration of the pins, each mechanical spotting
operation leaves a grid of spots spaced 4.5mm apart. To allow close
packing of spots (a typical center-to-center (CTC) distance is 250mm),
mechanical spotting operations are interleaved. The result is a
set of grids with 250mm spacing, often termed subgrids.
The set of subgrids in turn form a grid, called a metagrid.
If the CTC distance evenly divides 4.5mm, it is possible to create
an array with a single, large grid. However, designs of arrays
do not always allow such even divisibility. In addition, the replication
of subgrids allows the spot quantitation process to proceed more
easily, since the location of a small grid of spots can be done
easily, and the other subgrids can be found easily using the same
template.
The basic
proces
The basic process of printing an array depends on the arrayer.
Printing on a Cartesian PixSys arrayer is described here. The slide
platform holds 50 slides; it moves in the X direction. The robot
arm moves in the Y and Z direction. The arm and platform together
allow a pin to be positioned anywhere on and over the slide platform.
The platform and arm are covered with a humidity chamber, holding
its interior to 60% +- 5% humidity, to slow evaporation of the PCR
products or oligos in the spotting plate. A spotting operation
consists of washing the pins in nanopure water, dipping them in
the spotting plate wells for a second or so (to allow takeup into
the pin chamber), positioning the pins over each slide at the proper
position, and slowly touching the pins to the slide’s surface, depositing
the DNA in a coin-shaped spot.
Microarray
design
Design of spotting patterns in the form of metagrids and subgrids
is dependent on the number of plates to be spotted, the number and
configuration of the pins in the pin head, and the replication factor
for each spot. Most arrayers contain software to configure the
software to print a specified pattern, given the above parameters,
plus the CTC distance and subgrid dimension.
Sample tracking
Because the interleaving of DNA clones or oligos changes the ordering
of clones from the spotting plates to the slide, a tracking problem
is introduced. Tracking is often handled by the arrayer software,
but sometimes must be implemented within a lab in order to keep
track of lab-specific clone information and microarray layout designs.
A system for handling sample tracking conceptually involves the
identification of the spotting plates used in the printing, knowledge
of the microarray layout, and identification of each clone in each
spotting plate. Implementation can be done using spreadsheets and
a simple mapping program. In large production facilities, tracking
is often done by barcoding the spotting plates and slides, and accessing
a relational database containing the clone IDs for each spotting
plate.
Summary
Microarray construction requires careful design, which should take
into consideration the size of the gene or oligo set to be spotted
and the number of spotting pins available. It is a process which
requires extreme precision on the part of the operator. Design
of microarray printing technology is evolving at such a rapid pace
that the physical and chemical challenges in printing will be overcome
more quickly than the informatics challenge of tracking the clones
being spotted.
Construction
of a Microarray
This protocol describes the process of constructing a microarray.
It is assumed that the library or libraries to print have been chosen,
the microarray layout to accommodate these libraries has been designed,
and that there is a program for printing it on the Cartesian PixSys
arrayer. Layouts are chosen based on the number of plates to print,
how many replicate spots per clone are to be printed, whether an
additional control plate is to be printed, and the number of pins
available. Designing a new layout is beyond the scope of this protocol;
use Biodiscovery’s CloneTracker or other design software to do this.
Follow the
following procedure in constructing a microarray.
Choice of library (libraries) to print.If PCR spotting
plates have not been prepared from the library or libraries to be
spotted, prepare them using the PCR product preparation protocol.
Keep track of their PCR spotting plate ID, which gives information
about the bacterial source plate used, the date of preparation,
the quality of the individual PCR products in the plates, and the
dilution series and reaction number. If a control plate is to be
spotted, make a note of its ID as well.
Choice of slides.Choose 50 slides to be printed. Decide
the arrangement of slides on the slide platform. Label the slides
using a diamond-tipped marker with the slide position (row (1-10)
and column (A-E) on the platform, date of print, slide coating chemistry
(polylysine, silylyated, silanated, and manufacturer. Note that
the first 8-10 slides to be printed will not be useable because
of excess of DNA solution in the pins, so use cheaper slides in
this case.
Entry of information into logbook.Assign a microarray print
ID to the print run, and obtain the microarray pattern ID and program
name. The pattern ID gives a reference to information on the grid
layout and spotting order on the slide. Make an entry in the Microarray
Print Logbook, and enter this information, as well as the PCR spotting
plate IDs, control plate ID if any, and the slide information.
Also enter the date of print.
Online entry of information.There are two online logs (Excel
spreadsheets) to update: the Microarray Print Log and the Microarray
Slide Log. The Microarray Print Log has one entry per printing
operation; the Slide Log has an entry for each slide. In the Microarray
Print Log, enter the microarray print ID, pattern ID, date of print,
and the PCR spotting plate IDs for the library plates and the control
plate, if any. In the Slide Log, for each slide, enter the position
on the platform (B5, e.g.), slide chemistry, manufacturer, and lot
number or date of coating.
Placement of slides on platform. Wearing gloves, place
slides onto the platform in the designated positions. Use compressed
air to blow off any dust from the slides before placement. Open
the vacuum valves for all five columns of slides, and turn the vacuum
on to 5 lb pressure, to exert a slight pull on the slides. Snug
the slides to the left and bottom edges of their holders, then raise
the vacuum pressure to 12 lb.
Choice and testing of pins.The Telechem pins must constantly
be tested for spotmaking consistency. In addition, due to small
fluctuations in the pin head and pins, some pins will work better
in a given print head position than others. Choosing and testing
of pins before printing is therefore important.
- Follow Arrayer Startup directions below to start the
arrayer and its computer.
- Prepare a 384 well plate of PCR products that are used for
a test print as in the "Preparation of PCR spotting plate
for printing” protocol below . This should be an extra plate that
is not used on the arrays.
- Take out the slides in positions A1 and A2 on the platform.
Place test slides in those positions.
- Run the program “TestPinsProg01.ad”. It will prompt for the
pins to be loaded; do so and continue. Next it will prompt for
the spotting test plate to be placed in the holder. After the
plate is placed, continue with the program; it will preprint onto
A1 and print a 10x10 grid pattern on A2.
- After the program finishes, remove A2 and look at it under a
microscope to see if the spots are consistent and round. If a
pin or pins do not produce consistent, round spots, replace and/or
clean the underperforming pins. Clean using a solution of Telechem
ArrayIt Cleaning Solution in a sonicator; sonicate for only 5
seconds.
- Repeat Steps 5 and 6 until all of the pins are satisfactory.
When they are, then use this pin configuration. Record the pin
configuration into the Microarray Print Logbook beside the arrayer.
Running the array program.Start the array program chosen
above. It will assume that the Arrayer Startup procedure
has been done. It will prompt for spotting plates; prepare them
as in the preparation protocol and place them in the holder. While
one spotting plate is being printed, the next one can be initially
prepared by thawing it, spinning it down, and resealing the plastic
bag and placing it in the refrigerator (see the Preparation of
a PCR spotting plate for printing protocol below). When the
library plates have been printed, place the control plate into the
holder at the prompt.
Finishing
up.
Remove the last microplate and put it away after sealing (see
Storing PCR spotting plates after printing protocol below).
Turn the vacuum and humidifier of the Cartesian off .
Sometimes the humidity is high in the room and the spots do not
dry down under the humidity chamber. You must dry the spots by
placing the slides on a on flat heating block at 80-100c (setting
#4) for few seconds until moisture is just driven off. Bring the
heating block into the same room as the Cartesion to avoid dust
on the slides.
Run the “Home” program and very, very carefully unload the pins
and place them in the plexiglass storage rack.
Close the AxSys program window, turn off the arrayer, and release
the peristaltic pump heads. Empty the waste carboy.
Proceed to hydrate and crosslink the slides. immediately as in
the protocols below (also see the Protocol Inventory sheet labeled
Post Print Hydration and Crosslinking).
Post Print
slide treatment I: Hydrate the dried spots to make them more uniform:
___ 1. Pass dry slides, array side down, one-by-one over
hot water bath (70-90c) just until see fine mist (no big droplets)
over arrayed area.
____2. Place moistened slide on flat heating block at 80-100c (setting
#4) for few seconds until moisture is just driven off.
____3. Set aside to cool on a clean tray. Keep covered with foil.
Post Print
slide treatment II: UV cross-link the DNAs to the slide coating:
____1. Place dry slides, grid side up on a paper towel in StrataLinker
____2. Turn on power.
____3. Press 'Auto Cross Link'
____4. Set to 650 (=65mJ).
____5. Press 'Start'
____6. Beeps when finished. Press 'Reset' and turn power off.
____7. Store the slide is a dust free box labeled "Ready to Block".
Definition
of Terms
Pin configuration is the arrangement of pins in the arrayer
head. This includes a map of which pin is in which print head hole.
For example, a 2x2 configuration might look like:
meaning that pin 1 is in the lower left print head hole, 2 is
in the next one to the right, etc.
The slide platform on the arrayer holds 50 slides, in 5
columns (A-E) and 10 rows (1-10). A slide position on the
platform is designated A1, A2, …, A10, B1, …, E10.
The main slide coating chemistries used in the lab are:
poly-L-lysine, silanated, and silylyated. Varieties of slides include
Corning GAPS and Telechem. Poly-L-lysine slides are manufactured
in the lab (see Poly-L-lysine Slide Coating protocol) while
the others are ordered from various companies. It is important to
keep track of the . coating manufacture date in the case
of polylysine slides and the coating lot number in the case
of the company-manufactured slides.
Companion
protocols
These protocols accompany the protocol describing the construction
of a microarray.
Arrayer Startup
This procedure should be followed for any program to be run.
- Turn on the arrayer and computer. Close the peristaltic pump
heads.
- Check the program's certification status. All programs have
been tested using calibration pins and certified to work by the
programmer before being put into production. This is to prevent
damage to the pins from the misguiding of the robot arm and slide
platform caused by a programming error. Any change to the program
requires that it be recertified by a test run using calibration
pins. To check a program's certification, do the following:
- There is a log file of certified modifications to all microarray
programs in the Production_Programs directory, named
Production_Program_Log.txt. Open this log file and
search for the program being run. Note the date of the last
certified modification.
- Find the program in the Production_Programs directory.
In the View menu of the folder window, click Details
to get information on each program file in the folder. Note
the date under the Modified column. If this date is
later than the date found in the log, do NOT proceed -
the program was modified but not recertified. Tell the programmer
of this anomaly. If the Modified date is earlier than
the log date, then contact the programmer and inform him/her
that the log was not updated; let the programmer inform you
that it's safe to proceed. If the dates match, it is safe
to use the program.
- Prime the pumps by running PrimePumps.ad in the Production_Programs
directory (double click it and select the "Run" icon).
- Clean slide tray, source plate holder, wash basin, and head
with compressed air. Vacuuming is preferable.
- Have a supply of micropore filtered, deionized water available,
in the carboy marked "nanopure water". It should be less than
one month old. Attach supply tube to carboy. Supply tube is the
smaller tube, which feeds into the peristaltic pump on the left.
Be sure that the supply tube is well into the carboy.
- Empty waste carboy. Attach waste tube to carboy. Waste tube
is the larger one, feeding into the peristaltic pump on the right.
- Run "Armback.ad" to retract the arm to the back. Put on gloves
if not already done. Tape holes in the vacuum manifold that will
not be used by the pins.
- Turn on the humidifier. The humidifier should maintain 55%
to 65% humidity.
The arrayer is now ready for pin testing or a production run.
Preparation of a PCR spotting plate for printing
PCR spotting plates should be 384 well, Corning Costar PCR plates.
Each well should contain no more than 10 microliters. Spotting plates
are kept in the non-defrosting freezer, covered with foil film (3M
Scotch 425, core series 591) and enclosed in a resealable plastic
bag. Do the following to prepare a particular source plate for printing:
- Thaw source plates for 12 minutes.
- Spin plate using Jouan centrifuge at 2000 rpm for one minute.
- Carefully peel off the yellow tape label. There should be three
items on this label: (a) a PCR spotting plate ID, (b) a date of
creation of the spotting plate, and (c) the number of prints made
from this spotting plate. Increment the number of prints item
on the tape. Save this piece of tape if possible for reattachment
after printing.
- Carefully peel off the metallic adhesive covering.
- Place the source plate in the source plate holder on the microarray.
Be sure that the notched (A1) corner is at the bottom left.
Storing PCR spotting plates after printing
After the plate has been printed, remove it from the arrayer and
do the following.
- Cut a piece of foil film (3M Scotch 425, core series 591) so
that it covers the plate and extends at least 1/2 down all sides
of the plate.
- Seal the plate by running over the foil a blunt tipped object
(a Sharpie Regular Point works quite well).
- Add one to the number of prints on the yellow tape and reattach
it to the plate on top.
- Seal the plate in a plastic bag and store it in its assigned
position in the -20 C freezer.
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