Self-Digitization Microfluidic Chip for Absolute Quantification of mRNA in Single Cells

Abstract

Quantification of mRNA in single cells provides direct insight into how intercellular heterogeneity plays a role in disease progression and outcomes. Quantitative polymerase chain reaction (qPCR), the current gold standard for evaluating gene expression, is insufficient for providing absolute measurement of single-cell mRNA transcript abundance. Challenges include difficulties in handling small sample volumes and the high variability in measurements. Microfluidic digital PCR provides far better sensitivity for minute quantities of genetic material, but the typical format of this assay does not allow for counting of the absolute number of mRNA transcripts samples taken from single cells. Furthermore, a large fraction of the sample is often lost during sample handling in microfluidic digital PCR. Here, we report the absolute quantification of single-cell mRNA transcripts by digital, one-step reverse transcription PCR in a simple microfluidic array device called the self-digitization (SD) chip. By performing the reverse transcription step in digitized volumes, we find that the assay exhibits a linear signal across a wide range of total RNA concentrations and agrees well with standard curve qPCR. The SD chip is found to digitize a high percentage (86.7%) of the sample for single-cell experiments. Moreover, quantification of transferrin receptor mRNA in single cells agrees well with single-molecule fluorescence in situ hybridization experiments. The SD platform for absolute quantification of single-cell mRNA can be optimized for other genes and may be useful as an independent control method for the validation of mRNA quantification techniques.

A Self‐Digitization Dielectrophoretic (SD‐DEP) Chip for High‐Efficiency Single‐Cell Capture, On‐Demand Compartmentalization, and Downstream Nucleic Acid Analysis

“…To verify the performance of the SD‐DEP chip for single‐cell nucleic acid analysis, the expression of BCR‐ABL gene in K562 cells was measured. BCR‐ABL oncogene is a fusion gene resulting from a reciprocal translocation between chromosomes 9 and 22 and plays a critical role in the pathogenesis of chronic myeloid leukemia in humans, which is highly expressed in K562 cells . The cell lysis condition was optimized by performing bulk real‐time LAMP amplification of BCR‐ABL gene in K562 cells in LAMP reaction buffers without and with different concentrations of cell lysis agent (Triton‐X100), as shown in the Supporting Information, Figure S17.…”

Digital Nucleic Acid Detection Based on Microfluidic Lab-on-a-Chip Devices

“…To improve the efficiency of partitioning samples, a new generation of SD chip was invented with close to 100% efficiency in 2013 [74]. In 2014, based on the SD chip, digital RT-PCR was developed to absolutely quantify mRNA from single cells [75]. Due to the simplicity and robustness of the SD chip, the SD cdNAD is an inexpensive and easy-to-operate digital detection device.…”

Single cell digital polymerase chain reaction on self-priming compartmentalization chip

“…26 Compared with this method, we have developed two kinds of simpler and more efficient chips called self-priming compartmentalization (SPC) digital LAMP chip and integrated self-priming compartmentalization (SPC) digital PCR chip. 27,28 Both two kinds of chips have taken the advantage of the high gas solubility of poly(-dimethylsiloxane) (PDMS).…”