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Molecular Biology Protocols Mount
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12. Microarray-Based Analysis of Gene Expression
1. Oligonucleotide design: The DNAs to be printed as microarrays are
typically selected from a unique set of sequences from a particular species. Starting with expressed sequence tags (ESTs)
and/or cDNA sequences, we use TIGR Gene Indices Clustering Tool or PartiGene to
derive non-redundant clusters of unique sequences. Array Designer is then used to design
oligonucleotides of a specific length that possess maximum specificity for each
of those clusters, at the same time having a uniform range of overall binding
properties to facilitate hybridization under standard conditions. The oligos (50-mers) are synthesized by
Integrated DNA Technologies and are provided in 384-well plates at a
concentration of 250 μmol l-1.
2. Microarray printing: To 17 μl of
Pronto Universal Spotting Solution (Corning) in a 384-well plate, dispense 3 μl of oligonucleotide solution using a multipipette and
sterile filter tips. Mix well and
briefly centrifuge. Store at -20oC
between uses. Calibrate the OmniGrid
Accent arrayer to provide the density and spot pattern desired, following
instructions provided with the instrument.
Clean the slide trays and interior and exterior of the arrayer with a
moist paper towel and dry air to reduce dust.
Adjust chamber humidity to 62-64%.
Using gloves and without touching the printing surface, place slides
(Corning UltraGAPS) carefully into position on the slide trays, with bar code
oriented toward the left side of the arrayer.
Place 1-3 384-well plates
containing diluted oligos into position, wait 30 min for humidity to stabilize,
and start the printing protocol. Keep
the chamber closed as much as possible to maintain humidity control (and thus
spot size and morphology). When
printing is complete, dry the slides in a vacuum desiccator overnight,
then cross-link in a UV Stratalinker (Stratagene) at a setting of 6000 x 100 μJ. Store in a dust-free
environment in the dark.
As part of the arrayer calibration process, you must produce a
deconvolution file (GAL file) that relates the oligonucleotide ID to spot
location on the slide. This file will be
used by the scanner analysis software to track each spot’s data and is
produced by the “sample tracking” component of the arrayer program.
3. RNA reverse transcription and
labeling: High quality RNA is essential
for good microarray results. Check your
RNA extracts with the Agilent Bioanalyzer for quality and quantity! Follow the instructions provided with the
Promega ChipShot Direct Labeling kit, using Cy3 or Cy5 as the label, and
keeping the material in dim light or darkness during the process. Determine incorporation by analyzing 2 μl on a Nanodrop Spectrometer. Dry down the desired volume of each sample
with a SpeedVac centrifuge and resuspend in Pronto Short Oligo Hybridization
Solution (Corning).
4. Hybridization and washing: Follow instructions provided with the Pronto
Microarray Reagent System (Corning) and the MAUI Hybridization System
(BioMicro). Wherever indicated, dry
slides with filtered air (not canned “air”) provided by a
Microarray Air Jet (Arrayit) or equivalent.
5. Scanning: Load the appropriate GAL deconvolution file
(array list) from the arrayer software, found under the “file” icon
at the right of the GenePix screen.
Place the hybridized slide face down in the GenePix 4000B (Axon)
scanner, with the bar code toward the front of the instrument. Under the “hardware” icon at the
right of the screen, select “AutoPMT”. Check the box to automatically apply PMT
settings and start a data scan. Start
the scan, observing the process of locating blocks and spots. At the completion of each scan, check the
results visually. If features in a
specific block are not well aligned, highlight that block (icon or Ctrl+B) and
realign the features in that block (icon or F5). Repeat if necessary. Usually this is sufficient to obtain an
acceptable alignment. When you are
satisfied with the results, click the “Analyze” icon (or Alt+A). Then under the “file” icon, click
“Save Images” and “Save Results”, saving both in a
location that will be accessible to the analysis software.
6. Analysis: Using Acuity 4.0 (Axon) software, you may
analyze the microarray data in a variety of ways. Normalization is generally run initially,
followed by clustering, statistical analysis, and graphical displays of various
sorts. A detailed protocol is beyond the
scope of this introduction, but stay tuned!