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Obtaining reliable information from minute amounts of RNA using cDNA microarrays
Limei Hu1, Jing Wang, Keith Baggerly
1Cancer Genomics Core Laboratory-Department of Pathology, The University of Texas M D Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA. lhu@mdanderson.org
Background:
High density cDNA microarray technology provides a powerful tool to survey the activity of thousands of genes in normal and diseased cells, which helps us both to understand the molecular basis of the disease and to identify potential targets for therapeutic intervention. The promise of this technology has been hampered by the large amount of biological material required for the experiments (more than 50 microg of total RNA per array). We have modified an amplification procedure that requires only 1 microg of total RNA. Analyses of the results showed that most genes that were detected as expressed or differentially expressed using the regular protocol were also detected using the amplification protocol. In addition, many genes that were undetected or weakly detected using the regular protocol were clearly detected using the amplification protocol. We have carried out a series of confirmation studies by northern blotting, western blotting, and immunohistochemistry assays.
Results:
Our results showed that most of the new information revealed by the amplification protocol represents real gene activity in the cells.
Conclusion:
We have confirmed a powerful and consistent cDNA microarray procedure that can be used to study minute amounts of biological tissue.