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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). |
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.
Figures for Microarry Applications Using Promega Products
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Figure 1. Each quadrant on the slide contained spotted arrays as noted in the image. Each group was repeated until target ran out. Plates 1, 2 and 3 represent replicates of the identical purified material. |
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Figure 2. Image of one array using 532nm scan for Cy™3 label. Note the greater signal seen in the kanamycin (positive control) quadrants. |
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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 (red, lower row). |
