Researchers at Inje University in South Korea have developed a microfluidics-based chip that integrates three PCR needs into one device.
The chip, which is about the size of a microscope slide, performs mRNA extraction, cDNA synthesis, and gene amplification in a continuous manner, and may be suitable for performing genetic assays using small sample quantities, according to its developers.
Previous work by the group, led by Ki-Ho Han, created a device that performed sample extraction using lateral magnetophoresis with oligo-dT beads, followed by synthesis of cDNA.
In lateral magnetophoresis, a ferromagnetic wire array is placed over the entire area of an RNA microchannel at a certain angle with respect to the sample's flow. Applying an external magnetic field to the RNA microchannel forces the magnetic oligo-dT beads with bound mRNA to flow laterally into a DNA microchamber, with residue from the sample flowing into a waste chamber. In the DNA microchamber, bead-bound mRNA is mixed with cDNA synthesis reagents and PCR pre-mix, plus forward and reverse primers for selected genes. The cDNA is then synthesized by putting the chip on a hotplate,
The latest version of the chip, described in a paper published in January in RSC Advances, adds another microfluidics chamber where the beads are shuttled and RT-PCR is performed using a standard thermal cycler — a Life Technologies GeneAmp PCR System 9700, in this case.
The paper "is the first presentation of an on-chip RT-PCR microchip including three genetic functionalities for RNA extraction from a crude lysate sample, cDNA synthesis, and gene amplification," Han wrote in an email to PCR Insider.
"The on-chip integrated RT-microchip is fast and highly sensitive in comparison with typical genetic methods used daily in laboratories," Han said, adding that RNA extraction through to cDNA synthesis takes an hour and 11 minutes.
The integrated format of the group's chip is especially advantageous for RNA work because it "can prevent contamination and degradation by RNases from the outside environment," Han said. In addition, the researchers noted in their paper, the chip reduces the number and complexity of processing steps typically needed in conventional RT-PCR, making it conducive to use by relatively unskilled users, and the closed configuration protects users from being exposed to potentially infectious agents, making it suitable for infectious disease testing.
To evaluate the precision and range of applications of the new device, the authors tested it using whole blood, breast cancer cells, and throat swabs from patients.
From human blood lysate, they were able to amplify a 244-bp fragment of the human β-actin gene using as little as 0.1 microliters of finger-prick blood. They also amplified the cancer-related keratin 19 gene from the SKBR3 breast cancer cell line, and demonstrated detectable signal from a single cell.
Importantly, the team also tested whether the device could be used on clinical samples. Using Seegene's Seeplex RV7 kit for simultaneous detection of seven major respiratory viruses, they were able to isolate a number of different influenza genes from patient samples on-chip.
In their latest paper, Han and colleagues only demonstrated proof of principle for their fully integrated chip and did not detail the sensitivity of the device compared to conventional off-chip RT-PCR. However, in their previous iteration of the device, when gene amplification was performed externally using cDNA harvested from the microchip, sensitivity was higher than conventional RT-PCR methods.
For on-chip gene amplification, the researchers used the GeneAmp PCR System 9700 thermocycler from Life Technologies. They chose this platform because it was the one available to them, Han said.
Han and colleagues are currently looking for a company to help them commercialize the RT-PCR microchip. They have applied for US, European, and Korean patents for the lateral magnetophoresis method. Future directions also include further expanding the capabilities of the device. "We are going to integrate cell lysis functionality into the microchip as an additional sample preparation step," he said.