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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

ash-frozen tissue in a 1 ml microcentrifuge tube and vigorously homogenized for 30 sec using an Ultraturrax T8 homogenizer (Ika Laboratories). Total RNA was DNasetreated using DNA-free reagent (Ambion), and RNA yields were determined by fluorescence at 530 nm using RiboGreen RNA quantitation reagent (Molecular Probes) as described by the manufacturer. First-strand cDNAs were synthesized from equal amounts of total RNA (1 g/reaction) using oligo(dT) primers and SuperScript II reverse transcriptase (Invitrogen) according to the manufacturers instructions.

Gene-specific primers (GSPs) were designed to anneal near the 3' end of two mRNA transcripts and to generate PCR products 75300 base pairs long. Three GSP pairs amplify different overlapping regions of a single transcript that is enriched in the macula, while one GSP pair detects a human housekeeping gene transcript, β-actin (ACTB), which is constitutively expressed in the neural retina. The amplified regions spanned exon-exon junctions when possible. All primers were purchased from Proligo. RT-PCR was performed using the GSP pairs in reactions amplifying across a gradient of annealing temperatures to identify optimal reaction conditions for real-time RT-PCR, and PCR product lengths were verified on a 4.5% Super AcrylAgarose gel (DNA Technologies). Real-time quantitative RT-PCR was performed using an iCycler iQ system (Bio-Rad). The rate of accumulation of amplified DNA was measured by continuous monitoring of SYBR Green I (Molecular Probes) fluorescence. Melt curves of the reaction products were generated, and fluorescence data were collected at a temperature above the melting temperature of nonspecific products (Morrison et al. 1998).
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