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Q-RT-PCR: data analysis software for measurement of gene expression by competitive RT-PCR Peter A. Doris and Jon K. Hays, Sr.* Dept. of Cell Biology and Biochemistry and *Dept. of Computer Services Texas Tech University Health Sciences Center Lubbock, Texas 79430 Peter A. Doris, Ph.D. Dept. of Cell Biology and Biochemistry Texas Tech University Health Sciences Center Lubbock, TX 79430 email: cbbpad@wpoffice.net.ttuhsc.edu Contents: Abstract Introduction Theory Software Requirements Installation Use Notice Problem Reports References 2 ABSTRACT. We have developed software to assist in the computation of gene expression from data obtained in competitive RT-PCR. Here we describe the mathematical basis of competitive RT-PCR and discuss the criteria which must be met to permit accurate estimations of gene expression to be obtained using this technique. The software which has been developed assists in both the assessment of assay performance and in the routine analysis of data obtained in either titration-based or single tube quantitation of gene expression by competitive RT-PCR. The software is a 100Kb module which functions as a Microsoft Excel add-in. It is compatible with both Windows and Mac versions of Excel 5 and Excel 7 on the Windows 95 platform and employs the spreadsheet, statistical and graphing capabilities incorporated into Excel. 3 INTRODUCTION Quantitation of gene expression by competitive RT-PCR has been developed to the extent that it can now be considered, if applied rigorously, a well-evaluated, robust, quantitative method capable of providing accurate and precise estimates of the abundance of specific transcripts in RNA preparations, even in very small sample sizes (1). However, the mathematical concepts involved are complex and have not been conducive to the rigorous application of this technique. The software we describe here will, it is hoped, remove the problems associated with complex computations and allow the user to evaluate their competitive RT-PCR systems thoroughly and to perform routine data analysis easily. There are several requirements which can provide for quantitative accuracy in competitive RT-PCR. Many of these have been discussed in our papers (see references below). Some issues (RT efficiency differences between competitors and native template) have been studied and will be reported in forthcoming papers from our laboratory, please check Medline. The following assumptions are made in using this software: 1. The competitor used is a highly homologous RNA. We have not demonstrated that RT and PCR efficiencies can be identical for heterologous competitors (e.g. Clontech mimics). 2. The user has performed estimates using known starting quantities of both native and competitor RNA and has demonstrated that the system accurately est…