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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

ere extremely reproducible: macula-enriched target transcript CT in the macula-derived samples was 19.9 0.2 cycles (n = 11), and 21.4 0.3 cycles (n = 11) in the periphery-derived samples (compare Figures 2, 3, and 4A). Here we have demonstrated that the use of iQ supermix to optimize reaction conditions allows the best consistency and reproducibility from experiment to experiment.


Acknowledgements
We are greatly indebted to Drs Gregory J Riggins and Kathy Boon, Duke University Medical Center, Durham, NC, for their helpful expert advice. Supported in part by NEI R01 EY 11286, NEI P30 EY 05722 and a Career Development Award from Research to Prevent Blindness (CBR).


References
Boeckman F et al., Real-time PCR: General considerations, Bio-Rad bulletin 2593 (2001)

Bracete AM et al., Isolation of total RNA from small quantities of tissue and cells, Focus 21, 3839 (1999)

Loging WT et al., Identifying potential tumor markers and antigens by database mining and rapid expression screening, Genome Res 10, 13931402 (2000)

Morrison TB et al., Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification, Biotechniques 24, 954962 (1998)

The polymerase chain reaction (PCR) process is covered by patents owned by Hoffman-LaRoche. Use of the PCR process requires a license.

SYBR is a trademark of Molecular Probes, Inc.


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