Analysis of mRNA Manifestation in Fetal and Adult Murine Lacrimal Glands We next determined 0.01 and 0.01, respectively). Total RNA was isolated and analyzed for the manifestation of AQP family members using qPCR. The localization of AQPs in the adult lacrimal gland in adult murine lacrimal glands was also analyzed. Manifestation of Fluralaner mRNAs that were indicated at detectable levels were compared with those indicated in adult murine corneal and conjunctival cells. The intracellular localization of selected AQPs in the lacrimal glands was identified using immunohistochemistry. 2. Results 2.1. Manifestation of mRNAs in the Adult Murine Conjunctiva, Cornea, and Lacrimal Gland We compared the levels of mRNAs in the adult murine conjunctiva, cornea, and lacrimal gland. The manifestation levels of 0.01 and 0.01, respectively). 0.001 and 0.001, respectively). 0.01 and 0.01, respectively). The manifestation of 0.001 and 0.001, respectively). Significantly higher levels of 0.001 and 0.001, respectively). Open in a separate window Number 1 Analysis of manifestation of family members of mRNAs in an adult mouse lacrimal gland, conjunctiva, and cornea. Manifestation of each mRNA in an adult lacrimal gland (LG), conjunctival (CJ), and corneal (CO) cells was demonstrated. Total RNAs isolated from these three cells were subjected to qPCR analysis to detect the manifestation of (? 0.05, ?? 0.01, and ??? 0.001). 2.2. Analysis of mRNA Manifestation in Fetal and Adult Murine Lacrimal Glands We next selected 0.01 and 0.01, respectively). 0.01 and 0.01, respectively). 0.001 and 0.001, respectively). Open in a separate window Number 2 Analysis of family members of mRNA manifestation during the development of murine lacrimal gland. Manifestation of individual mRNAs ( 0.05, ?? 0.01, and ??? 0.001). mRNA manifestation in the murine lacrimal glands (fetal and adult), adult cornea, Fluralaner and conjunctiva were summarized in Table 1. mRNA expressions were relatively high in the adult lacrimal gland. Table 1 Summary of gene manifestation in murine lacrimal gland (E13.5d, E17.5d, and adult) and adult cornea and conjunctiva. published by the USA National Institutes of Health (NIH Publication No. 85-23, revised 1996) and were authorized by the Institutional Animal Care and Use Committee of Keio University School of Medicine (permission No. 08067-2). All animals were handled in full accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and institutional guidelines. C57BL/6 mice were purchased from CLEA Japan, Inc., Tokyo, Japan. Mice were sacrificed, and lacrimal gland tissues were removed from E13.5 (= 3; unknown gender) and E17.5 (= 3, unknown gender) embryos and adult male mice (8 weeks of age, = 3). Corneal and conjunctival tissues were also obtained from adult mice (= 3). Because embryo samples were too small, each quantitative qPCR was performed with the pool of 20 embryos, and 3 samples (= 3, 60 embryos) were analyzed in this study. For adult male mice, each sample was analyzed (= 3). For qPCR, samples were immediately treated with RNAlater? (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. For histopathological analysis, samples were immediately embedded in the optimal cutting heat (OCT) compound (Tissue-Tek; Miles Inc., Elkhart, IN) and stored at ?80C. 4.2. Quantitative Real-Time PCR (qPCR) Total cellular RNA from murine tissues was isolated using ISOGEN (Nippon Gene, Hilden, Germany) and purified using the RNeasy? Mini Kit (Qiagen) and DNase I (Qiagen) according to the manufacturer’s specifications. One microgram of total RNA was converted to cDNA (iScript cDNA Synthesis kit; BioRad) and used as a template. Quantitative real-time PCR was performed using SYBR? Green I, which binds double-stranded DNA, using a Step One plus (Life Technologies, Carlsbad, CA). The expression levels of mRNAs were normalized to the median level Fluralaner of a housekeeping gene (GAPDH). The copy number is usually expressed as the number of transcripts/ng total RNA. The primer sequences for murine and value of less than 0.05 was considered significant. Values are expressed as the mean standard?deviation (SD). Acknowledgments This research was supported by a Grant-in-Aid for Scientific Research (C) MEXT KAKENHI grant number 24592650 and the research grant for child health and development grant number 24-1 from the Ministry of Health, Labour and Welfare, Japan. Data Availability The gene and immunofluorescence data Fluralaner used to support the findings of Rabbit polyclonal to ZC3H12A this study are included within the article. Conflicts of Interest The authors declare that they have no conflict of interests or other interests that might be perceived to influence the results and/or discussion reported in this article. Authors’ Contributions NO and TK wrote the main manuscript text, and MK Fluralaner and MI prepared Figures ?Figures11?1C3. All authors reviewed the manuscript..