Abstractt
A number of articles have been published describing methods to produce fluorescent probes from RNA (or DNA) samples. These methods are conceptually similar. Broadly speaking, they involve some or all of the following procedures: template amplification, template transcription with concomitant incorporation of modified bases into the transcribed products (DNA or RNA), and signal amplification. The simplest technique relies on the direct incorporation of Cy3- or Cy5-labeled nucleotides into oligo-dT-primed cDNA by reverse transcriptase (RT). This method has proven to be reasonably robust, but it requires 50–200 μg of total RNA or 2–5 μg of poly(A) RNA per labeling (1–4). Thus, rather large tissue samples are required for each study.
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References
Duggan, D. J., Bittner, M., Chen, Y., Meltzer, P., and Trent, J. M. (1999) Expression profiling using cDNA microarrays. Nat. Genet. 21, 10–14.
National Human Genome Research Institute. Division of Intramural Research Microarray Project (uAP). http://www.nhgri.nih.gov/DIR/Microarray/main.html.
Schena, M., Shalon, D., Davis, R. W., and Brown, P. O. (1995) Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270, 467–470.
Stanford University. School of Medicine. The Brown Lab. The MGuide. http://cmgm.stanford.edu/pbrown/mguide/index.html.
Schroeder, B. G., Peterson, L. M., and Fleischmann, R. D. (2002) Improving quantitation and reproducibility in Mycobacterium tuberculosis DNA microarrays. J. Mol. Microbiol. Biotechnol. 4, 123–126.
Xiang, C. C., Kozhich, O., Chen, M., Inman, J. M., Phan, Q. N., Chen, Y., and Brownstein, M. J. (2002) Nat. Biotechnol. 20, 738–742.
Yu, J., Othman, M. I., Farjo, R., Zareparsi, S., MacMee, S. P., Yoshida, S., and Swaroop, A. (2002) Evaluation and optimization of procedures for target labeling and hybridization of cDNA microarrays. Mol. Vis. 8, 130–137.
Yue, H., Eastman, P. S., Wang, B. B., Minor, J., Doctolero, M. H., Nuttall, R. L., Stack, R., Becker, J. W., Montgovery, J. R., Vainer, M., and Johnston, R. (2001) An evaluation of the performance of cDNA microarrays for detecting changes in global mRNA expression. Nucleic Acids Res. 29, e41.
Stears, R. L., Getts, R. C., and Gullans, S. R. (2000) A novel, sensitive detection system for high-density microarrays using dendrimer technology. Physiol. Genomics 3, 93–99.
Karsten, S. L., Van Deerlin, V. M. D., Sabatti, C., Gill, L. H., and Geschwind, D. H. (2002) An evaluation of tyramide signal amplification and archived fixed and frozen tissue in microarray gene expression analysis. Nucleic Acids Res. 30, e4.
Van Gelder, R. N., von Zastrow, M. E., Yool, A., Dement, W. C., Barchas, J. D., and Eberwine, J. H. (1990) Amplified RNA synthesized from limited quantities of heterogeneous cDNA. Proc. Natl. Acad. Sci. USA 87, 1663–1667.
Wang, E., Miller, L. D., Ohnmacht, G. A., Liu, E. T., and Marincola, F. M. (2000) Nat. Biotechnol. 18, 457–459.
Hughes, T. R., Mao, M., Jones, A. R., et al. (2001) Nat. Biotechnol. 19, 342–347.
Mahadevappa, M. and Warrington, J. A. (1999) A high-density probe array sample preparation method using 10-to 100-fold fewer cells. Nat. Biotechnol. 17, 1134–1136.
Chen, Y., Kamat, V., Dougherty, E. R., Bittner, M. L., Meltzer, P. S., and Trent, J. M. Ratio statistics of gene expression levels and applications to microarray data analysis. Bioinformatics, in press.
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Xiang, C.C., Brownstein, M.J. (2003). Preparing Fluorescent Probes for Microarray Studies. In: Brownstein, M.J., Khodursky, A.B. (eds) Functional Genomics. Methods in Molecular Biology, vol 224. Humana Press. https://doi.org/10.1385/1-59259-364-X:55
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DOI: https://doi.org/10.1385/1-59259-364-X:55
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