Menu

Gene Expression in a Drop

Tens of thousands of individual cells can have their transcriptomes analyzed simultaneously thanks to two new techniques.

Aug 1, 2015
Ruth Williams

There are many reasons for analyzing the gene expression profiles of individual cells rather than a pooled population. But scaling up single-cell transcriptomics to analyze multiple cells in parallel has been challenging. “One can only analyze a few, or maybe 100 cells at a time,” says Donald Zack of Johns Hopkins Hospital in Baltimore.

“There’s a need for techniques that are unlimited in terms of the number of cells,” says Marc Kirschner of Harvard University. In some instances you need increased statistical power, he says. “But also, it’s often the case that you’re looking for a needle in a haystack”—that is, extremely rare cells.

Kirschner’s laboratory and that of Steve McCarroll, also at Harvard, have now independently broken the scale barrier for single-cell analyses. And surprisingly, the teams achieved their feats in similar ways despite only learning of each other’s approach well along in development. Both groups used microfluidic devices to create thousands of nanoliter-size aqueous droplets housing one cell and one set of barcoded primers each. The primers’ barcode sequences were unique to each droplet such that after reverse transcription and sequencing of the RNAs the researchers could identify the cells from which each transcriptome originated.

“The innovation was using an aqueous-oil emulsion,” says Zack, describing the oil-suspended droplets that essentially form hundreds of thousands, even millions, of tiny temporary test tubes. “Instead of needing a well or a little spot for each cell, now they could, by many orders of magnitude, increase throughput,” he says. Such droplets have been used for single-cell PCR, but the new techniques are the first to apply the technology to transcriptomics.

Kirschner’s team has used its technique, called inDrop, to study variations in seemingly uniform populations of mouse embryonic stem cells, while McCarroll’s team, using the Drop-seq approach it designed, has identified known and novel cell types in the mouse retina. As both researchers assert, however, the number of possible applications is vast. (Cell, 161:1187-1201, 2015 and Cell, 161:1202-14, 2015)

(1) Droplet formation: Primers, reagents (for lysing cells and performing reverse transcription), and a cell are packaged into an oil droplet. (2) Reaction: In the Drop-seq method (A), the cell is lysed and mRNA strands bind to barcoded primers stuck to a resin bead. The droplet breaks up and the RNA is then reverse transcribed.
The inDrop approach (B) uses a hydrogel microsphere to ferry reagents and barcoded primers into the droplet. After the cell is lysed, reverse transcription generates cDNAs with the tagged primers.
© GEORGE RETSECK
(3) Library construction: The cDNA is amplified to create an RNA library, then sequenced.© GEORGE RETSECK

TECHNIQUE BARCODED PRIMERS DROPLET PRODUCTIOJN AMOUNT OF CELLS SEQUENCED
Drop-Seq 16.8 million possible unique primers synthesized on surface of resin beads Millions per hour. About 1 percent contain both a bead and a cell. Many droplets contain a cell but no bead, so only a subset of cells is sequenced.
inDrop RNA sequencing 147,456 possible unique primers covalently linked to hydrogel microspheres Approximately 100,000 per hour. Nearly 100 percent contain microspheres and 10 percent also contain a cell. Roughly 90 percent of cells are captured and sequenced. Good approach for samples with limited cell numbers.

 

January 2019

Cannabis on Board

Research suggests ill effects of cannabinoids in the womb

Marketplace

Sponsored Product Updates

FORMULATRIX® digital PCR technology to be acquired by QIAGEN
FORMULATRIX® digital PCR technology to be acquired by QIAGEN
FORMULATRIX has announced that their digital PCR assets, including the CONSTELLATION® series of instruments, is being acquired by QIAGEN N.V. (NYSE: QGEN, Frankfurt Stock Exchange: QIA) for up to $260 million ($125 million upfront payment and $135 million of milestones).  QIAGEN has announced plans for a global launch in 2020 of a new series of digital PCR platforms that utilize the advanced dPCR technology developed by FORMULATRIX combined with QIAGEN’s expertise in assay development and automation.
Application of CRISPR/Cas to the Generation of Genetically Engineered Mice
Application of CRISPR/Cas to the Generation of Genetically Engineered Mice
With this application note from Taconic, learn about the power that the CRISPR/Cas system has to revolutionize the field of custom mouse model generation!
Translational Models of Obesity, Dysmetabolism, Diabetes, and Complications
Translational Models of Obesity, Dysmetabolism, Diabetes, and Complications
This webinar, from Crown Bioscience, presents a unique continuum of translational dysmetabolic platforms that more closely mimic human disease. Learn about using next-generation rodent and spontaneously diabetic non-human primate models to accurately model human-relevant disease progression and complications related to obesity and diabetes here!
BiochemAR: an augmented reality app for easy visualization of virtual 3D molecular models
BiochemAR: an augmented reality app for easy visualization of virtual 3D molecular models
Have you played Pokemon Go? Then you've used Augmented Reality (AR) technology! AR technology holds substantial promise and potential for providing a low-cost, easy to use digital platform for the manipulation of virtual 3D objects, including 3D models of biological macromolecules.