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D. Nguyen, H. Toshida, V. Vadlamudi, O. Akinyemi, R.W. Beuerman; Real-Time PCR Verification of Microarray Data in the Rat Lacrimal Gland Following Parasympathetic Denervation . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1024.
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Purpose: To verify by real-time PCR, gene expression analysis from microarrays of the rat lacrimal gland (LG) following preganglionic, parasympathetic denervation. Methods: Sprague-Dawley rats underwent unilateral pre-ganglionic parasympathetic denervation and sacrificed after 7 days. Total RNA was extracted from contralateral (Ctla) and denervated (Px) LG tissues (N=2), and used for microarray analysis. Sham denervated LG tissue and rat brain cDNA were used for comparison. The RT reaction was carried out using 1ug RNA. Semi-quantitative real-time PCR was carried out using either LUX (Light Upon eXtension) fluorogenic primers (common salivary protein (CSP), ribosome binding protein 1 isoform [mRRp47] or Sybr Green DNA binding dye (lipocortin, serum glucose-regulated kinase (SGK), oncomodulin) using the iCycler. All reactions were done either in duplicate or triplicate. Melting curve analysis was used as a quality control for specific product amplification. Mean cycle threshold (MCt) was determined and the T-test (p<0.05) was applied. Results: Microarray analysis showed a 7-fold upregulation for CSP and 3-fold downregulation for mRRp47. With real-time PCR, MCt values for CSP and mRRp47 in the Ctla were 37.00 and 29.08, respectively, and the sham values 37.95 and 29.34 were not significantly different. However, both were significantly different from the MCt value for the Px 33.18 and 30.03. CSP was not detected in the rat brain. The MCt values for lipocortin and SGK in the Ctla (23.9 and 27.59, respectively) and sham (24.1 and 26.67) were not significantly different, and both were significantly different from the Px (21.77 and 24.9). Microarray analysis found 4.4-fold and 2.8-fold increase in lipocortin and SGK expression, respectively. The MCt values for oncomodulin in the Ctla (19.28) and sham (20.02) were not significantly different; moreover, only the Ctla was significantly different from the Px (21.9). The microarray data showed a 2.6 fold decrease in oncomodulin expression.Conclusions: Array analysis has shown major changes in genes involved in immune regulation following parasympathetic denervation. In this study as the PCR results generally confirm the microarray analysis, we can develop a functional genomic scheme for the lacrimal gland. EY12416, RPB, EY002377, BRIN.
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