Real-time quantitative PCR (QPCR) has made quantifying mRNA transcripts, genotyping, verifying microarray data, and more a simple, automated process. But simple is a deceptive word and when it comes to QPCR, the devil is in the design details.
Primer and probe design, sample preparation, and normalization control selection are some common snares, says Greg Shipley, Director of the Quantitative Genomics Core Laboratory, University of Texas Health Science Center, Houston. While the urge may be to jump right to the cycler (see p. 68), "There's a lot of in silico homework," from determining whether your target has splice variants, to ensuring that it's specific.
The Scientist contacted several QPCR users to identify some of the issues they face and then consulted experts for solutions to their worries.
I-Hsiung Brandon Chen, formerly at the Scripps Research Institute in La Jolla, Calif., was using a Roche LightCycler 2.0 and SYBR Green chemistry to ...
