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D. Doyle, J.L. Loffreda, K.P. Mitton; Economical Q–PCR Kits for Mouse Retinal Gene Expression: ERI Eye Gene Matched Primer cDNA Kits . Invest. Ophthalmol. Vis. Sci. 2004;45(13):686.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The purpose was two–fold: 1) to develop economical quantitative PCR (real time) assays for mouse retinal genes expression that include matched primers/reference cDNA sets, and 2) to produce a companion database with practical information on reaction products and conditions. Methods: Several hot start Taq polymerases were compared for robustness with different primer sets. The gene expression Q–PCR kit development consisted of three parts: 1) computed evaluation of primer sets for intron/exon boundaries, literature errors, or custom design, 2) cloning of custom primers matched template cDNAs into pGemT Easy vector for use as standards, controls, and probes, and 3) experimental evaluation of annealing temperature ranges for each assay. Results: We have developed a collection of Q–PCR assays with companion standards and database for several retina specific genes. Those assays that have been developed are rhodopsin, PDE subunits alpha, beta, and gamma, blue opsin, green opsin, beta–actin, GAP–DH, Flt3, Fiz1, and NRL. We have found that custom assay conditions based on AmpliTaq Gold offers an economical advantage over pre–made kits. Conclusions: Q–PCR primer sets with matching standard cDNA were developed for measuring the expression of several retinal genes for use in the Eye Research Institute and will soon be available to the eye research community.
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