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Critical Reviews in Oral Biology & Medicine
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GENE EXPRESSION PROFILING BY DNA MICROARRAY TECHNOLOGY

Keisuke Iida and Ichiro Nishimura*

The Jane and Jerry Weintraub Center for Reconstructive Biotechnology, Division of Advanced Prosthodontics, Biomaterials and Hospital Dentistry, UCLA School of Dentistry, Box 951668, CHS B3-082, Los Angeles, California 90095-1668, USA;


Figure 1
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  		Figure 1. The number of published articles related to microarray technologies from 1993 to 2000. A rapid increase in the number of published articles from 1997 until the present is revealed by MEDLINE database searches.

 

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  		Figure 2. Principle of the cDNA microarray analysis system. Target cDNAs are cloned, and amplified by PCR. Purified PCR products are printed onto glass microscope slides with a robotic microarrayer. cDNA probes (test or reference) labeled with different fluorescent dyes (Cy3-dUTP and Cy5-dUTP) are synthesized from total RNA or mRNA derived from test and reference samples. Pooled probes are hybridized to the microarray. Hybridized fluorescent signals are detected with a dual-wavelength laser scanner. Separately scanned images are combined and pseudocolored by means of specialized computer software. Normalized ratios of Cy3/Cy5 are calculated for individual target genes.

 

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  		Figure 3. Computer-controlled custom-built (A) and commercially available (B) robotic microarrayers (UCLA microarray core). The entire device is surrounded by a clear plastic case so that the appropriate environment can be maintained. (C) A magnified picture showing a printing tip with a slot which can achieve passive dispensing of target genes by capillary reaction and direct contact to the solid surface. (D) A cluster of printing tips reduces the time of printing. (E) A magnified picture showing a cDNA microarray.

 

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  		Figure 4. cDNA microarrays following hybridization. (A) Typical cDNA microarray. (B) Inappropriate blocking procedure for microarray causes the comet tails.

 

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  		Figure 5. Hybridized fluorescent signal intensities vs. time. The decrease in the fluorescent signal intensities of the Cy dyes over time is demonstrated in both dyes. Although the intensities immediately after the hybridization and washing are the same levels for both Cy dyes, those at day 7 show different intensity due to a faster reduction of signal intensity in Cy5.

 

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  		Figure 6. Histograms representing the distributions of Cy3/Cy5 ratios. Continuous lines and interrupted lines indicate ± two-fold changes and 99% CIs, respectively. (A) A typical example of the "bell-shaped curve" distribution is shown. Note that the 99% CIs are included in the range of ± two-fold differences. (B) Comparison of dissimilar tissue shows deviated distribution. In this case, inconsistency is found between the ± two-fold differences and the 99% CIs.

 

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  		Figure 7. A scatter plot showing the results of comparative hybridization with the microarrays constructed with 96 duplicated housekeeping genes. (A) Comparison of RNA samples derived from the same origin indicates similar expression levels of housekeeping genes (Pearson's correlation coefficient, r = 0.94). (B) The result of microarray hybridization with RNA samples from dissimilar tissues reveals divergent transcriptional levels of the housekeeping genes (r = 0.70).

 

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  		Figure 8. A scatter plot comparing the expression ratios between mouse humerus and calvaria bones. Elevated gene expressions (> two-fold) of col1a1, col1a2, col9a1, col19a1, and osteonectin are found in calvaria compared with humerus.

 

Critical Reviews in Oral Biology & Medicine, Vol. 13, No. 1, 35-50 (2002)
DOI: 10.1177/154411130201300105
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