ABSTRACT
In molecular biology, the real-time polymerase chain reaction (real-time PCR), also called quantitative real-time PCR or kinetic polymerase chain reaction, is a laboratory technique that is based on the polymerase chain reaction (PCR), which is used to amplify and simultaneously quantify a targeted DNA molecule as well as mRNA. It enables both detection and quanti cation (as absolute number of copies or relative amount when normalized to DNA input or additional normalizing genes) of a speci c sequence in a DNA sample.1,2
9.1 Introduction .......................................................................................................................... 187 9.2 Protocols for Real-Time PCR and qRT-PCR ........................................................................ 188
9.2.1 Real-Time vs. Traditional PCR ................................................................................. 188 9.2.2 Limitations of End-Point PCR .................................................................................. 188 9.2.3 qRT-PCR Phases ....................................................................................................... 189 9.2.4 Quantitation .............................................................................................................. 189 9.2.5 Real-Time PCR Chemistries..................................................................................... 190
9.2.5.1 TaqMan Probes .......................................................................................... 190 9.2.5.2 Molecular Beacons .................................................................................... 190 9.2.5.3 Scorpions.................................................................................................... 190 9.2.5.4 SYBR Green .............................................................................................. 191
9.3 Procedures for RT-PCR ........................................................................................................ 191 9.3.1 Time Required .......................................................................................................... 191 9.3.2 Reagents and Equipment .......................................................................................... 191 9.3.3 Detailed Procedure ................................................................................................... 192
9.3.3.1 Reverse Transcription ................................................................................ 192 9.3.3.2 Real-Time PCR .......................................................................................... 192 9.3.3.3 Troubleshooting ......................................................................................... 193
9.3.4 PCR Optimization: Reaction Conditions and Components ..................................... 193 9.3.5 Protocol for Competitive RT-PCR to Quantify mRNA ........................................... 194
9.3.5.1 Making Internal Standard RNAs ............................................................... 194 9.3.5.2 Quantitation of mRNA .............................................................................. 194
References ...................................................................................................................................... 195
This procedure follows the general principle of the PCR (Chapter 3). Its key feature is that the ampli ed DNA is quanti ed as it accumulates in the reaction in real time after each ampli cation cycle. The two common methods of quanti cation are as follows:
• The use of §uorescent dyes that intercalate with double-stranded DNA • Modi ed DNA oligonucleotide probes that §uoresce when hybridized with a complemen-
tary DNA
Frequently, the real-time PCR is combined with reverse transcription (RT) in order to quantify messenger RNA in cells or tissues.3 In order to detect and quantify gene expression from small amounts of RNA, ampli cation of the gene transcript is necessary. The PCR is a common method for amplifying DNA. For mRNA-based PCR, the RNA sample is rst reverse transcribed to cDNA with reverse transcriptase. Gene-speci c primers are then utilized to amplify the target gene with increased PCR cycles.4,5
Development of PCR technologies based on reverse transcription and §uorophores permits measurement of DNA ampli cation during PCR in real time; that is, the ampli ed product is measured at each PCR cycle.6,7 The data that are generated can be analyzed by computer software in order to calculate relative gene expression in several samples or mRNA copy number. Real-time PCR can also be applied to the detection and quanti cation of DNA in samples in order to determine the presence and abundance of a particular DNA sequence in these samples.8-11
Real-time chemistries allow for the detection of PCR ampli cation during the early phases of the reaction. Measuring the kinetics of the reaction in the early phases of PCR provides a distinct advantage over traditional PCR detection. Traditional methods use agarose gels for the detection of PCR ampli cation at the nal phase or end point of the PCR reaction.
