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R. M. Corrales, C. S. De Paiva, W. J. Farley, B. A. S. Castillon, M. E. Stern, S. C. Pflugfelder; Absolute Quantitation of Cytokine and Transcription Factors in the Normal Murine Ocular Surface. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5901.
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To determine the absolute quantity of target nucleic acid sequences of T helper (Th)-1, Th-2, Th-17 transcription factors and signature cytokines in healthy murine corneal and conjunctival epithelium samples.Material and
Total RNA was isolated and the first-strand cDNA was synthesized from corneal and conjunctival epithelia that was collected and pooled from 10 eyes of C57BL6 mice aged 6-8 weeks. Absolute real time PCR was performed using a Taqman Fast Universal PCR Master Mix and specific Taqman Probes to determine the absolute quantity of the transcription factors GATA-3, RORT and T-bet as well as the cytokines IL-2, IFN-, IL-2, IL-4, IL-6, IL1-2a, IL-13, IL-17A, IL-17F, and IL-21. The PCR products were quantified by comparing the corneal and conjunctival samples with standard curves made with samples of known quantities (from 107 to 1 copies/µl). Three replicates of each sample and each dilution point in the standard curve were done. The results are presented as the mean of 3 separate experiments. The t test was used for statistical analysis.
The conjunctiva has significantly higher number of transcriptions factors transcripts than cornea (p<0.005, <0.0005, and p=0.00005 for GATA-3, RORT and T-bet respectively). GATA-3 is the predominant Th transcription factor in the normal conjunctiva (3800 copies/µl), followed by RORt (1300 copies/µl). T-bet was the lowest (40 copies/µl). In the cornea, RORt was the predominant transcription factor detected (900 copies/µl) with low levels of the GATA-3 and T-bet (10 and 4 copies/µl, respectively). Both tissues have similar Th signature cytokines, except for IL-13 and IL-6, which were the significantly higher (p<0.05, and <0.01, respectively) in the normal conjunctiva.
Conjunctiva had greater levels of Th transcription factors and signature cytokines than cornea. Th1 transcription factor were at barely detectable levels in both tissues. There was a mix of Th-17 and Th-2 transcription factors in conjunctiva, and Th-17 transcription factor predominance in cornea. IL-17A had the highest levels among the Th signature cytokines in conjunctiva.
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