PCR-based methods for the detection of changes in gene expression are powerful techniques. Traditional quantitative RT-PCR analyzes differences in gene expression by comparing samples with equal starting amounts of RNA. After optimizing for the number of cycles (and other parameters), the products are separated on a gel, and compared with an internal control. While this method is good, products are seen after the exponential phase of amplification, which lowers the sensitivity.
Real-time PCR is an improvement on regular
1. Products are measured after each cycle, rather than at the end of the run. This eliminates the need for determining the optimal cycle number (all PCRs are run for 40 cycles).
2. A standard annealing temperature is used, eliminating the need for optimization. Nearly all primers are designed to work at this temperature.
3. Depending on the dye used, the sensitivity is increased by several orders of magnitude.
4. Samples are analyzed during the exponential phase, where differences in quantity of products are maximized.
This web page describes two standard methods (SYBR Green and TaqMan) for real-time PCR, starting with cell culture or animal tissue. Sample preparation, reverse transcription, primer design, materials needed, and other methods are covered. Analysis of data will also be described.
However, this site doesn't cover the very basics, such as what is PCR, what is a primer, etc... For that kind of basic information, please see the Wikipedia article.
The goal of this webpage is to get you started in using this relatively simple technique for analyzing gene expression. I provide the recipes, supply list, and everything else you need to get started! Why use this page? It is a practical guide to getting started and includes recipes that was originally designed as an internal instruction guide for my laboratory at Rutgers University.
Disclaimer: This guide is a basic guide. There are many nuances to doing PCR that will not be covered here. The information on this web page is for informational purposes only. Use at your own risk. The author(s) assume NO RESPONSIBILITY for any errors on this page. Joshua P. Gray is an Assistant Professor at the United States Coast Guard Academy in New London, CT. The methods and opinions described here are his own, and do not necessarily represent those of the U.S. Coast Guard or of the U.S. government.
ABOUT THE AUTHOR
Joshua P. Gray is an Assistant Professor of Chemistry at the United States Coast Guard Academy in New London, CT. His research interests include sulfur mustard (mustard gas), type I diabetes, and oxidative stress. [link]