Integrating whole transcriptome assays on a lab-on-a-chip for single cell gene profiling
Lab Chip. 2008-01-01; 8(3): 443
DOI: 10.1039/b716543a
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1. Lab Chip. 2008 Mar;8(3):443-50. doi: 10.1039/b716543a. Epub 2008 Jan 31.
Integrating whole transcriptome assays on a lab-on-a-chip for single cell gene
profiling.
Bontoux N(1), Dauphinot L, Vitalis T, Studer V, Chen Y, Rossier J, Potier MC.
Author information:
(1)Laboratoire de Neurobiologie et Diversité Cellulaire, CNRS UMR 7637, ESPCI,
Paris Cedex 05, France.
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.
DOI: 10.1039/b716543a
PMID: 18305863 [Indexed for MEDLINE]