Biomedical Engineering Reference
In-Depth Information
Real-Time Quantitative RT-PCR
for mRNA Profiling
Stephen A. Bustin
1
and Tania Nolan
2
1
Institute of Cell and Molecular Science, Barts and The London, Queen Mary's School of Medicine
and Dentistry, University of London, Whitechapel, London, UK
2
Sigma-Aldrich House, Haverhill, Suffolk, UK
6.1 Introduction
The real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) [1]
is characterized by its specificity, sensitivity, simplicity and speed. These attributes have
made it the method of choice for the detection and quantification of RNA [2, 3], and have
effected its extensive use in biotechnology [4], microbiology [5], virology [6] and molecular
medicine [7] applications. The assay involves a conventional reverse transcription (RT)
procedure, followed by the qPCR step, which makes use of fluorescent reporter molecules
to combine the amplification and detection components of the PCR in a single tube format
[8, 9]. An increase in fluorescent signal is proportional to the amount of DNA produced
during each PCR cycle and produces a characteristic threshold cycle (
C
t
) or crossing point
(
C
p
) for every reaction. The
C
t
or
C
p
is defined as the PCR cycle at which the signal first
rises above background fluorescence. The more target there is in the starting material, the
earlier the instrument can detect the fluorescence and the lower the
C
t
. This correlation
between fluorescence and amount of amplified product permits accurate quantification of
target molecules over a wide dynamic range and the homogeneous format significantly
reduces hands-on time and the risk of contamination [10].
The consistency and reliability of RT-qPCR assays depend on the proper execution of
a number of steps, in particular those relating to sample selection, template quality, assay
design and data analysis [11], as well as the correct application of statistical models and
methods for data analysis [12]. If correct procedure is adhered to, then results are gener-
ally robust, reproducible and accurate. Specifically, the reliability of the RT-qPCR assay is
