SYBR® Green and TaqMan® quantitative PCR arrays: expression profile of genes relevant to a pathway or a disease state

Abstract

Quantitative PCR arrays are the most reliable and accurate tool for analyzing the expression of a focused panel of genes relevant to a pathway or a disease state. PCR arrays allow gene expression analysis with the sensitivity, dynamic range, and specificity of a real-time PCR as well as the multi-gene profiling capability of a microarray. Differences among real-time PCR kits used in PCR arrays are largely restricted to the DNA polymerases and the detection methods used. In this chapter, we provide a step-by-step protocol for the two detection methods most commonly used in PCR arrays, known as SYBR(®) Green and TaqMan(®), which are based on two different approaches to detect PCR products. While SYBR(®) Green uses a binding dye that intercalates nonspecifically into double-stranded DNA, the TaqMan(®) approach relies on a fluorogenic oligonucleotide probe that binds only the DNA sequence between the two PCR primers. Therefore, only specific PCR product can generate a fluorescent signal in TaqMan(®) PCR. Here we also provide a comparison of the SYBR(®) Green and TaqMan(®) approaches and highlight their advantages and disadvantages to help the user to choose the best platform.