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Rapid, Reproducible Real-Time Quantitative RT-PCR Using the iCycler iQ Real-Time PCR Detection System and iQ Supermix, Rev A

Jessica N Ebright1 and Catherine Bowes Rickman1, 2, 1Departments of Ophthalmology and 2Cell Biology, Duke University Medical Center, Durham, NC 27710 USA

Correspondence: Catherine Bowes Rickman, PhD, Department of Ophthalmology, Duke University Medical Center, Box 3802, Durham, NC 27710, Phone (919) 668-0648, Fax (919) 684-3687, E-mail: bowes007@duke.edu


Introduction
We are interested in identifying genes that are differentially expressed within the central (macular) region of the human retina. Expression profiles of thousands of genes from this small, highly specialized region of the central nervous system were obtained by comparative screening of human cDNA microarrays with human macula- and mid-peripheral retina (periphery)-derived RNAs. In order to validate the maculaenriched expression of genes identified by our array analysis, we must rely on a PCR-based method of quantitation. Realtime RT-PCR quantitates the initial amount of a template with more specificity, sensitivity, and reproducibility than any other method. There are many factors that contribute to the consistent performance of a real-time quantitative RT-PCR assay, and many aspects that must be optimized when putting this powerful technology to work in a new experimental system.


Methods
Total RNA was isolated from 4 mm trephine punches of neural retina from two areas, the macula and the midperiphery, using Trizol reagent (Invitrogen) with glycogen added as a carrier as described by Bracete et al. (1999) with the following modification: 0.9 ml Trizol reagent plus 13.5 l glycogen (20 mg/ml, Roche Molecular Biochemicals) was added to fl
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