Chapter 16: A Concrete Case: A Microfluidic Device for Single Cell Whole Transcriptome Analysis
Published:18 Oct 2010
N. Bontoux, L. Dauphinot, and M. Potier, in Unravelling Single Cell Genomics, ed. N. Bontoux, M. Potier, L. Dauphinot, H. Craighead, H. Kroto, and P. O'Brien, The Royal Society of Chemistry, 2010, ch. 16, pp. 243-260.
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Single cell whole transcriptome analysis, i.e. the analysis of all the genes that are expressed by a cell at a given time and under given physiological or pathological conditions, constitutes a major challenge in understanding cellular diversity and the complexity of living organisms. Indeed, such analyses will be key in unravelling cellular regulatory networks and understanding cell growth, differentiation and migration mechanisms.1 They are also of significant interest for diagnosis and could prove a very efficient tool to identify new therapeutic targets.2
With the recent development of DNA microarrays, the transcriptome, i.e. the expression of all the genes, can now be studied in a single experiment. However, current labelling and detection methods require a starting amount of total RNA of about 100ng, which is around 104 times more than the content of a single cell. The sensitivity thus undoubtedly needs to be improved to achieve accurate single cell whole transcriptome analysis. In this context, microfluidic devices offer interesting perspectives since they enable studies to be performed at the pico or nanoliter scale.
In this chapter, we will detail our microfluidic approach for whole gene profiling of single cells. We will briefly review the choice of protocols for single cell transcriptome amplification as well as materials and techniques that can be used to fabricate microfluidic devices. We will describe the integration of the reverse-transcription (RT) and polymerase chain reaction (PCR) steps on chip and then discuss how all the steps of the biological protocol can be integrated in a single lab-on-a-chip.