Real‐Time Quantitative RT‐PCR for mRNA Profiling

Abstract

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 generally robust, reproducible and accurate. Specifically, the reliability of the RT-qPCR assay is Genomics: Essential M ethodsEdite d by Mike S ta r ke y a nd R a mna th Ela swa r a pu

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