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Focus: DNA Microarrays
Microarray Applications Using Promega Products
The aim of these experiments was to
demonstrate the utility of Promega products in DNA microarray applications. We tested the
Wizard® MagneSil™ PCR Clean-Up System (Cat.# A1930),
ImProm-II™ Reverse Transcription System (Cat.# A3800)
and SV Spin Columns (included in Wizard® Plus SV Minipreps System Cat.# A1330). |
By Randy Hoffman, B.S.
Promega Corporation
Introduction
Here we demonstrate the utility of Promega products in DNA microarray applications. We
tested Wizard® MagneSil™ PCR Purification
System(a) (Cat.# A1930) for removal of reaction contaminants post-amplification,
ImProm-II™ Reverse Transcription System (Cat.# A3800)
for labeled probe synthesis and SV Spin Columns
(Cat.# A1330)
for probe purification. Arrays of RT-PCR products were produced at the University of
Wisconsin Gene Expression Center, Genome Center of Wisconsin and University of Wisconsin
Biotechnology Center.
Results
To test Promega products in microarray research, a model system was developed in which
kanamycin RT-PCR products, produced using the ImProm-II™ R.T.
System, were spotted onto poly-L-lysine-coated slides. The final
array consisted of RT-PCR products full-strength as well as dilutions of 1:5, 1:25, 1:100
purified with MagneSil™ PCR Clean-Up System. As a positive control, we screened the
array with fluorescent probes derived from kanamycin mRNA transcripts. Probe derived from b-actin RT-PCR product was used as a negative control. Slides
consisted of samples arrayed in 2 x 2 factorial quadrants of target (b-actin and kanamycin) by purification method (MagneSil™ PCR
Clean-Up and another purification reagent). Figure 1 shows the spotting map for
each quadrant and Figure 2 shows a
representative quadrant.
MagneSil™ PCR Clean-Up purified products were visible after hybridization with the
kanamycin fluorescent probes. RT-PCR product diluted 1:100 is clearly visible (Figure 3).
For background control, we spotted 3X SSC, and the average relative pixel intensity for
these spots was subtracted from those for all positive test spots. Also, faint b-actin background was seen in the negative control quadrant (Figure 2).
Discussion
Wizard® MagneSil™ PCR Clean-Up System is an easy and efficient method
to purify PCR or RT-PCR products prior to microarray spotting. Moreover, it allows
significant dilution of spotting material when many arrays are needed from the same
starting material. ImProm-II™ Reverse Transcriptase-generated fluorescent probes
performed well in this experiment. Earlier work has shown incorporation efficiencies
similar to or better than SuperScript™ II. Of special note, the Cy™5 probe gave
signals as high as the Cy™3 probe. The combination of the SV Spin Basket and SV
Neutralization Buffer included in the Wizard® Plus SV
Minipreps DNA Purification System (Cat.#
A1330) provide for easy and quick probe purification.
Acknowledgments
We acknowledge the expert advice of Sandra Splinter and David Frisch at the UW Gene
Expression Center as well as array production and post processing by Sandra Splinter,
David Frisch and Matt Lawler.
(a)The PCR process is covered by patents issued and
applicable in certain countries. Promega does not encourage or support the unauthorized or
unlicensed use of the PCR process.
*Products may be covered by pending or issued patents. Please visit
our patent and trademark web page for more information.
Wizard is a trademark of Promega Corporation and is registered with the U.S. Patent
and Trademark Office. ImProm-II is a trademark of Promega Corporation.
Cy is a trademark of Molecular Probes. SuperScript is a trademark of Life
Technologies, Inc.
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Figure
1. Each quadrant on the slide contained spotted arrays as noted in the image. |
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Figure 2. Image of one array using
532nm scan for Cy™3 label. |
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Figure
3. Montage of quadrants scanned at 532nm for Cy™3 label (green, upper row) and
635nm for Cy™5 label... |
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