| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
From the Centers for Disease Control and Prevention, United States Public Health Service, Department of Health and Human Services, Atlanta, Georgia
Before gene expression profiling with microarray technology can be transferred to the diagnostic setting, we must have alternative approaches for synthesizing probe from limited RNA samples, and we must understand the limits of reproducibility in interpreting gene expression results. The current gold standard of probes for use with both microarrays and high-density filter arrays are synthesized from 1 µg of purified poly(A)+ RNA. We evaluated two approaches for synthesizing cDNA probes from total RNA with subsequent hybridization to high-density filter arrays: 1) reverse transcription (RT) of 5 µg total RNA and 2) RT-polymerase chain reaction (RT-PCR) of 1 µg total RNA, using the SMART system. The reproducibility of these two approaches was compared to the current gold standard. All three methods were highly reproducible. Triplicate experiments resulted in the following concordance correlation coefficients to evaluate reproducibility: 0.88 for the gold standard, 0.86 for cDNA probe synthesized by RT from total RNA, and 0.96 for the SMART cDNA probe synthesized from total RNA. We also compared the expression profile of 588 genes for the total RNA methods to that obtained with the gold standard. Of 150 positive genes detected by the gold standard, 97 (65%) were detected by cDNA probe synthesized by RT of total RNA, and 122 (81%) were detected by the SMART cDNA probe. We conclude that SMART cDNA probe produces highly reproducible results and yields gene expression profiles that represent the majority of transcripts detected with the gold standard.
This article has been cited by other articles:
 |
|
 |
|
  M. Fluck, C. Dapp, S. Schmutz, E. Wit, and H. Hoppeler Transcriptional profiling of tissue plasticity: role of shifts in gene expression and technical limitations J Appl Physiol, August 1, 2005; 99(2): 397 - 413. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Grau, X. Sole, A. Obrador, G. Tarafa, E. Vendrell, J. Valls, V. Moreno, M. A. Peinado, and G. Capella Validation of RNA Arbitrarily Primed PCR Probes Hybridized to Glass cDNA Microarrays: Application to the Analysis of Limited Samples Clin. Chem., January 1, 2005; 51(1): 93 - 101. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. E. Rogler, T. Tchaikovskaya, R. Norel, A. Massimi, C. Plescia, E. Rubashevsky, P. Siebert, and L. E. Rogler RNA expression microarrays (REMs), a high-throughput method to measure differences in gene expression in diverse biological samples Nucleic Acids Res., August 25, 2004; 32(15): e120 - e120. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Pellis, N. L. W. Franssen-van Hal, J. Burema, and J. Keijer The intraclass correlation coefficient applied for evaluation of data correction, labeling methods, and rectal biopsy sampling in DNA microarray experiments Physiol Genomics, December 16, 2003; 16(1): 99 - 106. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Fink, S. Kohlhoff, M. M. Stein, J. Hanze, N. Weissmann, F. Rose, E. Akkayagil, D. Manz, F. Grimminger, W. Seeger, et al. cDNA Array Hybridization after Laser-Assisted Microdissection from Nonneoplastic Tissue Am. J. Pathol., January 1, 2002; 160(1): 81 - 90. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. S. Rajeevan, S. D. Vernon, N. Taysavang, and E. R. Unger Validation of Array-Based Gene Expression Profiles by Real-Time (Kinetic) RT-PCR J. Mol. Diagn., February 1, 2001; 3(1): 26 - 31. [Abstract] [Full Text]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
