Methods and platforms for analysis of nucleic acids from single-cell based on microfluidics

Abstract

Single-cell nucleic acid analysis aims at discovering the genetic differences between individual cells which is well known as the cellular heterogeneity. This technology facilitates cancer diagnosis, stem cell research, immune system analysis, and other life science applications. The conventional platforms for single-cell nucleic acid analysis more rely on manual operation or bulky devices. Recently, the emerging microfluidic technology has provided a perfect platform for single-cell nucleic acid analysis with the characteristic of accurate and automatic single-cell manipulation. In this review, we briefly summarized the procedure of single-cell nucleic acid analysis including single-cell isolation, single-cell lysis, nucleic acid amplification, and genetic analysis. And then, three representative microfluidic platforms for single-cell nucleic acid analysis are concluded as valve-, microwell-, and droplet-based platforms. Furthermore, we described the state-of-the-art integrated single-cell nucleic acid analysis systems based on the three platforms. Finally, the future development and challenges of microfluidics-based single-cell nucleic acid analysis are discussed as well.

Microbial Single-Cell Analysis: What Can We Learn From Mammalian?

“…Generally, genotypic analysis in microbes includes analyzing DNA and RNA among individuals by revealing the composition and quantification of sequences. For the performance of each methods, our previous work and other reports have summarized the state-of-the-art sequencing methods for microbial single-cell analysis in detail, interested readers could refer to these articles ( Chen et al, 2017b ; Kaster and Sobol, 2020 ; Liu et al, 2021 ). To our knowledge, the main obstacle in this field is to amplify the total DNA from one microbe, but it has been basically solved in the past decade ( Chen et al, 2017b ; Kaster and Sobol, 2020 ; Liu et al, 2021 ).…”

“…For the performance of each methods, our previous work and other reports have summarized the state-of-the-art sequencing methods for microbial single-cell analysis in detail, interested readers could refer to these articles ( Chen et al, 2017b ; Kaster and Sobol, 2020 ; Liu et al, 2021 ). To our knowledge, the main obstacle in this field is to amplify the total DNA from one microbe, but it has been basically solved in the past decade ( Chen et al, 2017b ; Kaster and Sobol, 2020 ; Liu et al, 2021 ). For example, by using the mainstream MDA amplification method ( Spits et al, 2006 ), researchers have successfully achieved the amplification with 10 fg of Corynebacterium glutamicum DNA with a digital microfluidic device, within only 2 h to reach the amount required for successful sequencing ( Liu et al, 2021 ).…”

“…Simultaneously profile phenotype (like metabolites with Ramanome) and genotype (genome or transcriptome) from the same cell could be very helpful in some specific fields, such as finding the related gene clusters for producing specific natural products ( Xu et al, 2020 ; Jing et al, 2021 ). In addition, the state-of-the-art techniques for single-cell analysis prefer to use microfluidics as the bridge between phenotypic sorting and genotypic analysis ( Liu et al, 2021 ). Using microfluidics, individual cells are sorted into small oil-water particles, together with the regents for cell lysis and further genotypic analysis, which both avoids possible contamination in common tube-based experiments, and improves the reaction efficiency since the concentrations of both nucleic acids and enzymes are raised ( Chen et al, 2017b ; Liu et al, 2021 ).…”

“…In addition, the state-of-the-art techniques for single-cell analysis prefer to use microfluidics as the bridge between phenotypic sorting and genotypic analysis ( Liu et al, 2021 ). Using microfluidics, individual cells are sorted into small oil-water particles, together with the regents for cell lysis and further genotypic analysis, which both avoids possible contamination in common tube-based experiments, and improves the reaction efficiency since the concentrations of both nucleic acids and enzymes are raised ( Chen et al, 2017b ; Liu et al, 2021 ). In summary, we supposed that an ideal microbial single-cell analysis platform should be high-throughput and composed of both phenotypic sorting and genotypic analysis, with most of the processes being conducted in microfluidics to achieve the best performance ( Figure 1 ).…”

“…In addition, microbiologists could use single-cell level methods to uncover the hidden information in microbes without requiring axenic cultivation, which could greatly expand the scope of target microbes ( Stepanauskas, 2012 ). Here, since several articles have successfully reviewed current methods being applied in microbes ( Chen et al, 2017b ; Kaster and Sobol, 2020 ; Liu et al, 2021 ), we briefly highlighted recent progress on methods for single-cell analysis, including phenotypic and genotypic analysis, especially on those methods which only proved their performance in mammalian cells but showed potentials for microbial applications, and presented perspectives on the future trends of technology development in the field of analyzing microbes at single-cell level.…”