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

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 complementar...

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Published in:Lab on a chip 2008-01, Vol.8 (3), p.443-450
Main Authors: Bontoux, N, Dauphinot, L, Vitalis, T, Studer, V, Chen, Y, Rossier, J, Potier, M-C
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cited_by cdi_FETCH-LOGICAL-c281t-94b55567ef28264201491bb32aaf6ae30e17bd53c6e4f4d402fbc3037f3024f83
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creator Bontoux, N
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description 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.
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source Royal Society of Chemistry:Jisc Collections:Royal Society of Chemistry Read and Publish 2022-2024 (reading list)
subjects Animals
Base Sequence
Brain - metabolism
DNA Primers
Gene Expression Profiling
Mice
Miniaturization
Polymerase Chain Reaction
RNA, Messenger - genetics
title Integrating whole transcriptome assays on a lab-on-a-chip for single cell gene profiling
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