NDLI: Integrating whole transcriptome assays on a lab-on-a-chip for single cell gene profiling

Content Provider	Royal Society of Chemistry (RSC)
Author	Rossier, J. Chen, Y. Studer, V. Vitalis, T. Bontoux, N. Dauphinot, L. Potier, M-C.
Copyright Year	2008
Abstract	To correlate gene expression profiles to fundamental biological processes such as cell growth, differentiation and migration, it is essential to work at the single cell level. Gene expression analysis always starts with the relatively low efficient reverse transcription (RT) of RNA into complementary DNA (cDNA), an essential step as unprocessed RNAs will not be analysed further. In this paper, we present a novel method for RT that uses microfluidics to manipulate nanolitre volumes. We compare our method to conventional protocols performed in microlitre volumes. More specifically, reverse transcription was performed either in a polydimethylsiloxane (PDMS) rotary mixer or in a tube, using a single cell amount of mouse brain RNA (10 pg), and was followed by a template-switching PCR (TS-PCR) amplification step. We demonstrate that, using the microfluidic protocol, 74% of the genes expressed in mouse brain were detected, while only 4% were found with the conventional approach. We next profiled single neuronal progenitors. Using our microfluidic approach, i.e. performing cell capture, lysis and reverse transcription on-chip followed by TS-PCR amplification in tube, a mean of 5000 genes were detected in each neuron, which corresponds to the expected number of genes expressed in a single cell. This demonstrates the outstanding sensitivity of the microfluidic method.
Starting Page	443
Ending Page	450
Page Count	8
File Format	HTM / HTML PDF
ISSN	14730197
Volume Number	8
Issue Number	3
Journal	Lab on a Chip
DOI	10.1039/b716543a
Language	English
Publisher	Royal Society of Chemistry
Access Restriction	Open
Subject Keyword	Polymerase chain reaction Reverse transcriptase Neuron Complementary DNA Mouse Lysis Litre Gene expression Polydimethylsiloxane Microfluidics DNA sequencing
Content Type	Text
Resource Type	Article
Subject	Chemistry Nanoscience and Nanotechnology Biochemistry Bioengineering Biomedical Engineering

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