Quantitative Real-time PCR, a Tutorial
by Joshua Gray, Ph.D. - Assistant Professor, Rutgers University

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 PCR. 
   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. 

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!   

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.