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MEETING NEWS Rajendrani Mukhopadhyay reports from MicroTAS 2005—Boston, Mass.
Finding all the mRNA in a single cell
NATHALIE BONTOUX
The cDNA is then amplified by PCR by valves,” explains Bontoux. “When mRNA molecules pass along the necesin a microfuge tube and analyzed by you actuate the valves, there is pumping sary information from DNA to the sites conventional gel electrophoresis. With in the loop and so you [can] mix reof protein synthesis to give a cell its their device, Bontoux and colleagues agents.” The channels of the device are identity. “If you want to characterize a can detect transcripts spanning the coated with bovine serum albumin to cell at a given time, you have to look at range from 100 bp to >6 kb. the mRNA,” says Nathalie (a) (b) “We now can amplify all Bontoux, a graduate stusizes of mRNA. If you do dent working with Jean the same reaction in a tube, Rossier of the Ecole Supériyou amplify only short traneure de Physique et de Chiscripts,” states Bontoux. mie Industrielles and Yong The main difference beChen at the Centre Nationtween the reaction taking al de la Recherche Scienplace in a tube and in the tifique (both in France). device is the RNA concentraBontoux and colleagues are tion. When reverse transcripdeveloping a microfluidic tion is performed in a microdevice to analyze all the fuge tube, the final reaction mRNA molecules in a sinvolume is 10 µL; the final regle cell. 1 2 3 4 5 action volume in the device Analyzing mRNA is easiis ~8 nL. Because the amount er said than done, because (a) The rotary microfluidic device for reverse transcription can produce of RNA remains at 10 pg, it constitutes 6 kb. (b) In contrast, reverse transcription the RNA is ~1000 more total RNA in a cell. The in the conventional microfuge tube reaction is limited to 100-bp to ~2.5-kb concentrated in the device sizes of the mRNA tranfragments. Lanes on the gel are (1) 100-bp DNA ladder, (2) Hind III DNA than in a tube. Bontoux says, scripts span a range from ladder, (3) reaction done in a microfuge tube, (4) reaction done in the mi“That’s probably what ex100 bp to >6 kb. Although crofluidic device, and (5) PCR negative control. plains the better results we >5000 genes are expressed get because, for a low amount of RNA, prevent nonspecific adsorption of in a cell at any given time, existing mothe reaction efficiency is concentrationreagents and samples to the walls. lecular biology techniques can track dependent.” The reverse-transcription The investigators mix reagents inside only ~100 genes from a single cell. Curreaction is also quicker in the microfluthe device for a reaction called reverse rent mRNA detection methods are inidic device (20 min) than in the tube transcription. In this reaction, which inadequate for researchers like Bontoux (several hours). cidentally is used by certain types of and colleagues, who would like to idenBontoux and colleagues are hoping viruses during infection, the sequences tify all the mRNA in single neuronal to integrate PCR with the reverse-tranencoded in the mRNA molecules are cells and correlate the gene expression scription reaction in future designs of converted into a format called completo the highly diverse characteristics of the device. Bontoux says, “If you don’t mentary DNA (cDNA). The device is neurons. take your cDNA out of the device, you first filled with 10 pg of RNA, equivaTo develop an alternative mRNA delent to the RNA content of a single cell, have less chance of losing some sample tection method, the investigators are or to have any kind of contamination.” and the different reagents for reverse creating a rotary microfluidic device out She adds that PCR may benefit from transcription. It is kept at 1 °C to preof PDMS by using the multilayer soft vent the reaction from taking place. The the device. “We have proven [that relithography technique developed by verse transcription] works better beStephen Quake’s group at Stanford Uni- investigators initiate mixing in the ring cause [the RNA] is more concentrated. and heat the device up to 37 °C so that versity. The device has two layers—one contains the channels, and the other acts the reaction can take place. The reaction We may also expect the PCR to work better and require fewer cycles.” The inis allowed to run for 20 min before it is as the valves and pumps. “Our device is vestigators would also like to incorpocooled back down to 1 °C. The device [shaped] like a ring. On this ring, there rate a detection method for the ampliis then flushed with water to collect the are two inlet channels as well as outlet fied cDNA in their device. cDNA produced by the reaction. channels. All these channels are closed 6
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