Multiplex PCR is an extremely enabling molecular biology technique, allowing the amplification of multiple targets in a single PCR experiment. There are a broad range of research applications for multiplex PCR, including pathogen identification, high-throughput SNP genotyping, gene expression profiling, template quantitation, RNA detection and even forensic studies.
Gene expression profiling of CTCs
The presence of CTCs (circulating tumor cells) in peripheral blood has been linked to various types of cancer, e.g., breast, colorectal and prostate cancer (1). A variety of molecular assays have been developed for CTC detection and molecular characterization.
Molecular assays for studying gene expression on CTCs are based on the sensitivity and specificity of real-time RT-PCR (real-time reverse transcription PCR). Multiplex PCR enables the generation of richer gene expression profiling data by amplifying many gene-targets using a very small amount of precious CTC-derived nucleic acids. Multiplexed gene expression analysis assays give researchers an efficient and robust method to perform mRNA expression analysis of multiple genes by real-time RT-PCR on small numbers of CTCs and even allows molecular characterization for a single CTC. Considering that multiplex real-time RT-PCR offers a unique advantage for the detection and molecular characterization of CTCs, it is an important tool that can be used to expand our understanding of metastasis and potentially improve patient management.
Multiplexing – Efficient and simple! What about setup?
Once up and running, multiplex reactions are efficient, cost-effective and simple to run, but the optimization of a multiplex PCR reaction is not trivial (2). There are challenges to overcome. Each individual reaction must be optimized before it is added to the multiplex reaction – then the individual reactions must all work when combined. The optimization of each reaction usually takes several attempts to develop a robust and efficient multiplex PCR reaction (3).
If you would like to find out more about Critical factors and how to overcome challenges in multiplex PCR, join my webinar on Thursday, 31 August at 15:30 EST.
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- 1. Cristofanilli, M., Budd, G.T., Ellis, M.J., 2004. Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N. Engl. J. Med. 351, 781–791. (link)
2. Bustin (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction. J. Mol. Lab. Endocrinology 25, 169–193 (link)
3. Markoulatos. et al. (2002) Multiplex Polymerase Chain Reaction: A Practical approach. J. Clin. Lab. Anal 16, 47–51 (link)