Application Story: Dr Winston Patrick KuoWinston Patrick Kuo carries out research at the Department of Genetics in the research group of Dr. Constance L. Cepko, in conjunction with the Decision Systems Group, and the Department of Oral Medicine, Infection, and Immunity, all at Harvard Medical School, Boston, MA. Here he describes his research and how the introduction of ProbeLibrary™ has benefited his work.
1. What is your research area?
In our group, we are mainly focused on the development of the central nervous system of vertebrates, with an emphasis on the development of the retina, where we use both the mouse and chick animal models. The laboratory has studied the expression patterns of retinal cells over various time points using several genomic approaches including serial analysis of gene expression (SAGE). My study initiated by comparing this approach to that of hybridization-based methods using the same samples. Currently, I am engaged in a large-scale cross-platform study evaluating many aspects of gene expression based technologies. The two main samples in the study are mouse cortex and retina samples for which we have SAGE libraries. The goal of my study is to develop “high quality” data using a systematic approach in an attempt to integrate data across different platforms reducing the need to duplicate experiments, thus reducing cost and time. Currently I am comparing the performance of 14 different gene expression based technologies. Some these technologies include: Affymetrix GeneChips®, Agilent, Agilix, Amersham, Applied Biosystems, Academic cDNA, Illumina, Mergen, Lynx (MPSS), Operon, and SAGE.
2. Why did you choose to test ProbeLibrary™ - what were your needs?
In our group we have always used SYBR® Green for reasons of cost. However, comparing these different gene expression based technologies generates an enormous amount of data that would require biological validation by real time PCR. When using SYBR® Green we use publicly available design software to find the optimal primers. However, primer tests and assay optimization are very time consuming and we have often had difficulties in reproducing our results. Therefore, I was looking for a cost-effective and a highly efficient probe based system that would provide highly reproducible results in a semi-high throughput fashion given the nature of this study. We decided on the ProbeLibrary system after a small in-house study.
3. What have been the main benefits of the ProbeLibrary™?
Two things: reduced assay development time, resulting in increased throughput; and improved reproducibility! The ProbeFinder™ assay design software is very reliable and we basically find little, if any, need for optimization. The assay failure rate is below 5% and we can reproduce our results in a way that was impossible before. The ProbeLibrary™ has really enabled high-throughput real time PCR in our lab.
4. Does ProbeLibrary give you additional advantages/opportunities?
The ProbeFinder™ assay design software is very flexible and informative. We can map everything to a particular region of a transcript and easily carry out cross validating assays, because several assays are available for the same transcripts. It is very important to have this flexibility since all the mappings across the platforms have been based on exon-mapping. Having a reliable and flexible real time PCR system such as ProbeLibrary enables us to carry out whole transcriptome profiling in a cost effective manner. Additionally, the genome-wide coverage of the ProbeLibrary is very advantageous for this type of study.
5. When will we hear more about your microarray platform study?
I will be presenting results of the study at several upcoming conferences, one of which will be at the Beyond Genome conference in San Francisco in June as part of the “Panel Discussion of Validation of Microarray Data” session and early fall where I will Chair of the” Diagnostic Applications -Validated Microarray Case Studies for Human Diseases and Biomarker Discovery” session. Furthermore, I expect to publish the results during 2005.
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