Methods for Comparing Across-Target Sequences in Small-Scale qRT-PCR Experiments

• Activity Number: 125
• Type: Contributed
• Date/Time: Monday, August 2, 2010 : 8:30 AM to 10:20 AM
• Sponsor: Biometrics Section
• Author(s): Stuart W. Gardner*+ and Jack M. Gallup and Max Morris and F. Chris Minion
• Companies: Iowa State University and Iowa State University and Iowa State University and Iowa State University
• Keywords: Quantitative real-time reverse transcription PCR (qRT-PCR) ; PREXCEL-Q ; Delta Method Variance Estimates ; Top down versus Bottom Up Statistics

Abstract

Quantitative real-time reverse transcription PCR is the top validating technology for gene expression-capable "next generation" technologies. However, high-throughput technologies are still largely cost-prohibitive. Thus there is a need for strategic design of a limited number of qRT-PCR plates to generate quality data for meaningful statistical inference. This study compares the PREXCEL-Q-based "Stock I" idea to a design that uses serial dilutions of each individual sample. We demonstrate that by using a limited number of plates, both designs generate equivalent, meaningful and statistically valid results for small-scale qRT-PCR. A novel method to compare the relative quantity estimates of several different targets is presented along with various methods to obtain error estimates of fold change and relative quantity by applying both top down and bottom up statistical approaches.