PAX6 mRNA Transcript Analysis in Various Ocular/Non-Ocular Tissues

Abstract

PAX6 is a critical regulatory gene located on human chromosome 11p13, is expressed in many ocular tissues, and non-ocular tissues like brain, and pancreas. The expression of mRNA transcript has been investigated in this study since PAX6 is likely to play a vital role in the development of eye. RNA was extracted from various ocular tissues including lens epithelium, retina, iris, non-ocular tissues Trabecular Meshwork cells (TM cell line), HeLa cells. Besides the mutant and wild type blood cells were also included and total RNAs were subjected to convert in to Complementary DNA (cDNA). The high sensitive reverse transcriptase polymerase chain reaction (RT-PCR) was performed to monitor the mRNA transcript level to detect the expression profiles of the ocular/non-ocular tissues taken for this study. The competitive RT-PCR analysis was carried out using the house keeping gene beta actin as a control in both the ocular and non-ocular tissues. The level of beta actin expression in ocular/non-ocular tissues was equal and showed 540bp amplification. Subsequently the PAX6 mRNA transcript was analysed in these tissues. The expression level of PAX6 was observed in the ocular tissues such as retina, iris and lens epithelium which expressed approximately 600, 855, 1013 and 1055bp amplification. In the non-ocular HeLa cells the PAX6 expression was abnormally high and showed approximately 1.2kb amplification. No PAX6 expression was observed in the wild type WBC cells; however the mutant WBC amplified an irrelevant transcript. Sequencing the irrelevant transcript revealed the homology hint to various chromosomes particularly of hypothetical LOC441763 mRNA. RT-PCR amplification confirms the presence of various PAX6 mRNA expressions in ocular and non-ocular tissues. Lens epithelium, retina and iris tissues have significant PAX6 expression while the retina contains an overwhelming majority of mRNAs encoding crystallins and other genes but still express PAX6 isoforms at high levels. HeLa is a fibroblast based cell line and it would not expect a fibroblast to express a lot of PAX6 However PAX6 gene expression might be high in these cells for some reason. The PAX6 gene partial expression was found in mutant WBC and not in wild type WBC cells, the real fact for this reason is unknown. PAX6 mutations should be analyzed with effective functional assays, which will be useful for future study.