Wednesday, November 13, 2002
Generating Microarrays for Retinal Studies Using a
High-throughput DNA Isolation System
Macular Degeneration is
a blinding disease that afflicts over 10 million people in the United States,
and 30% by the time they are 75 years old. At the moment, we have a poor
understanding of its causes and no effective treatments (1). To further our
knowledge of the progression of this disease, we have generated a custom
microarray starting with 9,216 sequenced mouse retinal cDNAs (2). One of the
most critical steps in microarray generation is the isolation of high-quality
plasmid DNA as a substrate for PCR amplification. After considering several
options, we chose the Concert™ 96 system to isolate high-copy number plasmid DNA
using the 96-well plate format. The system produces high-quality DNA in 45-60
minutes. In addition, the DNA performed well in subsequent steps of microarray
production.
Methods
Figure 1A
shows the process for generating a cDNA microarray.
Starting material.
A non-redundant panel of
9,216 sequenced mouse retinal cDNAs was used as starting material for microarray
generation.
\
Plasmid isolation.
DH10B cells containing
plasmid DNA were grown in 96-well plates in 1.25 ml 2XYT medium containing 50
µg/µl of ampicillin. The wells were inoculated by sterilizing a pin replicator
with 100% ethanol and transferring frozen stocks of each clone from a 384-well
plate. After growing overnight at 37°C (18-22 hours) at 250 rpm, each plate was
harvested using Protocol B of the Concert™ 96 Plasmid Purification System (Cat.
no. 12263-018, Invitrogen, Carlsbad, CA). Cells were harvested and resuspended
in 200 µl Cell Suspension Buffer. They were transferred to a filter plate
sitting atop a receiver plate containing 200 µl isopropanol per well. Cells were
lysed with 100 µl Lysis Buffer containing RNase A for 10 min. at room
temperature. The stacked plates were spun at 3,000 x g for 15 min at 4°C. The
filter plate was discarded and the filtrate poured off. The receiver plate was
briefly dried with a stack of paper towels. Each well was washed with 200 µl 70%
ethanol and incubated at room temperature for 2 min. The liquid was again poured
off and the receiver plate spun briefly inverted to 35 x g. The plate was then
allowed to air dry for 5 min. Pellets were resuspended in 25 µl TE buffer.
PCR.
PCR was performed in a
96-well format utilizing 100-µl reactions. The PCR master mix consisted of 50 mM
KCl, 10 mM Tris-HCl (pH 8.3), 2mM MgCl2, 0.01% gelatin, 0.2 mM dNTPs, 1 µg each
primer and 2.5 units Promega
Taq
Polymerase (Promega,
Madison, WI). The primers were M13/pUC forward (CGC CAG GGT TTT CCC AGT CAC GAC)
and M13/pUC reverse (AGC GGA TAA CAA TTT CAC ACA GGA). Template DNA was 3 µl of
Concert™ 96-isolated plasmid. PCR reactions were heated at 94°C for 4 min.,
followed by 40 cycles of 94°C for 30 s., 58°C for 30 s., and 72°C for 1.5 min.
in a 96-well plate. The final extension was 4 min. at 72°C.
Microarray generation.
PCR products were purified
and run on a 1% agarose gel stained with ethidium bromide (Figure 1B).
After transferring the clones back into a 384-well format,slides were printed by
the Yale Keck Facility on in-house poly L-Lysine coated slides utilizing a
GeneMachines Omnigrid robotic arrayer (GeneMachines, San Carlos, CA) (Figure 1C,
page 18).
Microarray analysis.
Microarrays were hybridized
with probes made from mouse retina using the Genisphere cDNA Array Kit (Genisphere
Inc, Hatfield, PA). Slides were scanned on a GenePix 4000A scanner (Figure
2) and the data manipulated with GenePix software (Axon Instruments, Union City,
CA).
Results
and Discussion
Microarray analysis is a complex process that is prone to
technical difficulties if reagents and input material are not of suitable
quality. Our lab has found that the quality of the input plasmid DNA can
dramatically affect results. Any data that is to be analyzed downstream by
microarray analysis must start with quality plasmid DNA. The Concert™ 96 Plasmid
Purification System is a good choice for our microarray project because of the
efficiency of the system and the quality of the DNA produced. In isolating 9,216
clones, the reproducibility of high throughput DNA isolation was imperative for
our work. Since the Concert™ 96 system procedure contains no mixing or vacuum
filtration steps, cross contamination was held to a minimum. This was judged at
a gross level by sequence analysis of a random selection of clones and at a
finer level by PCR amplification and gel analysis. Our retinal microarrays have
already given insights into changing patterns of gene expression during
embryonic development. In addition to detecting the appearance of known genes at
expected times in development, we have found numerous novel genes that show
similar expression patterns and may thus be expressed by the same cell types or
used in the same pathway. The microarrays are also helping us understand changes
that can occur in retinal aging.
Genes whose
expression changes markedly during aging are good candidates for risk factors
for degenerative diseases such as Macular Degeneration