Elaborating Lab-on-a-Chips for Single-cell Transcriptome Analysis
Nathalie Bontoux, Luce Dauphinot and Marie-Claude Potier
from: Lab-on-a-Chip Technology (Vol. 2): Biomolecular Separation and Analysis (Edited by: Keith E. Herold and Avraham Rasooly). Caister Academic Press, U.K. (2009)
Working at the single cell level is becoming incontrovertible in many fields of biology particularly when studying gene expression in complex tissues such as the brain. Gene expression protocols always start with the conversion of RNA to complementary DNA (cDNA) by reverse transcription. This low efficiency reaction is crucial since unconverted RNAs will not be analyzed further. In this chapter, a detailed protocol for single cell whole transcriptome analysis is presented. This protocol includes a novel microfluidics step for high yield reverse transcription performed in devices made of polydimethylsiloxane (PDMS). These devices allow the manipulation of nanoliter volumes, thus increasing the concentration of starting RNAs. This methodwas validated by comparing it to conventional protocols performed in microliter volumes using single cell amount of mouse brain RNA (10 pg). Single gene PCR was then integrated to the reverse transcription reaction on the same PDMS device in a separate chamber. The template switching PCR reaction for whole transcriptome amplification was, however, performed in conventional tubes since the yield was very poor in microfluidics devices because of molecular crowding. Gene profiling of single neuronal progenitors is discussed at the end of the chapter. Using this microfluidic approach (cell capture, lysis and reverse transcription in the microfluidic device followed by template switching 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 that was developed read more ...