NEW YORK (GenomeWeb) – Researchers at Zhejiang University in Hangzhou, China, have demonstrated the utility of an automated microfluidics handling robot to print nanoliter-sized droplets on a reusable chip for single-cell RT-qPCR.
The technique is "simple to use and compatible with currently used biological operations and systems" which should make it "easily accepted by [the] biological research community," Qun Fang, a professor of analytical chemistry and co-author of a Scientific Reports paper describing the method, told GenomeWeb in an email.
The new system is an improvement over conventional methods, which can include manipulating single cells under the microscope and are often low throughput and consume copious reagents. It is also more flexible and less expensive than commercial products for similar purposes, such as single-cell analysis arrays from Fluidigm, Fang said.
In the method, a microfluidics robot prints a dilute cell suspension on hydrophilic spots of a silicon chip to create an array. Droplets can be between 60 picoliters and 200 nanoliters, and evaporation in this semi-open system is limited by using a layer of mineral oil.
Cells are compartmentalized in the droplets based on Poisson distribution. "There are zero, one, two, three, and more cells per droplet [and] the highest probability for single-cell encapsulation in droplets was about 35 percent," Fang noted.
The droplets are identified based on their spatial information, and number of cells distributed in each droplet is measured and, Fang said. Then, all the procedures required for single-cell RT-qPCR can be performed in the droplets, including thermal lysis, reverse transcription, and quantitative PCR with fluorescence detection.
In the study, the method was used to measure expression levels of mir-122 in the Huh-7 hepatocellular cell line.
Specifically, an array of 360 2-nanoliter droplets had 126 droplets containing single cells, and empty droplets had Ct values beyond the cutoff value. The copy numbers of mir-122, a liver-specific micro RNA, were highly variable, from about 3,000 to almost 80,000, with a mean copy number comparable to reported values for Huh-7 cells. RT-qPCR required overcoming cell lysate-induced PCR inhibition by increasing concentrations of PCR reagents.
The current single-cell-distributing system is based on the group's sequential operation droplet array, or SODA system, published previously in Analytical Chemistry.
"The SODA system is an automated and flexible microfluidic platform for picoliter-scale droplet manipulation, analysis, and screening using the programmable combination of [a] capillary-based liquid aspirating-depositing operation and moving of the oil-covered 2D droplet array," Fang explained.
Fang's group originally developed the SODA system due to its need for a "versatile and automated microfluidic platform for biological research" that didn't require "complicated and long-term microchip fabrication and system building for each specific application," he said.
In terms of operation flexibility, system versatility, and cost, the technique could ultimately be competitive with Fluidigm's single-cell platforms, Fang said.
"The major advantage of the SODA droplet robot technique is flexibility in performing multiple liquid handing operations — it can achieve various liquid handling steps including droplet assembling, generation, indexing, transferring, splitting, and fusion in the picoliter range by using the same hardware and different software programs," he said.
Besides single-cell gene expression analysis, the group has also used the SODA system for chemical library screening and cell-based drug studies.
"Now we are developing a digital PCR system based on the SODA technique. It is quite practical in current biological research, because one such platform can deal with multiple biological applications," Fang said.
Since Fluidigm launched an early-access program for its C1 Single-Cell AutoPrep System for single-cell RT-qPCR in 2012, that platform has become a major revenue driver for the company. The C1 had a total installed base of 1,325 instruments at the end of 2014, and recently-previewed new products for the system included a chip to prepare up to 800 cells for RNA-seq.
Fang said his group now has a patent in China on the SODA technique and is working on an international patent.
"We have no collaboration in industry yet [but] we have a strong willingness to commercialize our technique [and] now we are seeking commercial partnerships," he said.