Single Cell RNA Sequencing (scRNA-seq)

RNA sequencing is a genomic approach used to detect and quantitatively analyze messenger RNA (mRNA) within a biological sample. RNA-seq is used to study cellular responses in relation to the microenvironment and offers the ability to study cellular characteristics like differentiation, proliferation, or tumorigenesis. RNA-seq, however, is usually conducted on samples composed of thousands to millions of cells, meaning that this technology really assesses the interaction of a population of cells. Alternatively, single cell RNA-seq (scRNA-seq) allows for direct transcriptome and heterogeneity analysis between individual cells within a highly diverse population thereof.

What is Single-cell RNA Sequencing?

---

scRNA-seq focuses on the biology of individual cells amongst each other which has, among other things, enabled cellular gene expression to be evaluated with incredible resolution. By evaluating cells on a cell-by-cell basis, scRNA-seq has helped in deciphering fundamental information about gene expression, splicing patterns, gene co-expression, and genetic regulatory networks.

 

scRNA-seq has been used to understand transcriptional differences between cells, which has a number of applications in advancing medicine. scRNA-seq can be used to identify rare cell populations, like malignant tumor cells or hyper-responsive immune cells, that may have otherwise gone unnoticed with other techniques. scRNA-seq can be used to analyze heterogeneous cell states and trace the lineage and developmental relationships between each. In research, scRNA-seq has been used to help de-elucify the roles particular cells have in embryonic development, cancer differentiation, and lymphocyte diversification.

 

How scRNA-Seq Works

---

 First, cells within a biological sample must be isolated and captured. Traditionally, this was performed by limiting dilution assays or micromanipulation. Though a step forward at the time, both methods are time-consuming, low throughput, and have significantly low accuracy and precision.

 

Today, many techniques exist to easily isolate individual cells. For example, flow-activated cell sorting (FACS) is a specialized method of flow cytometry that separates cells using fluorescence technologies. Another method, magnetic-activated cell sorting (MACS), uses a magnetic field to separate and isolate individual cells. Both FACS and MACS are preferred isolation techniques when the target cell may be sparse amongst a complex sample, or if target cell expression is low. Other notable approaches to isolating single cells from a complex biological sample include microdissection, microfluidic platforms, and droplet-based methods.

If preferred, individually identified cells can then be barcoded. Molecular barcodes are short nucleotide tags that are used to identify sequences, or reads, that originate from the target cell. Numerous single cell barcoding technologies are available, including barcoded magnetic beads. These barcoded beads act similar to other magnetic bead technologies and allow for effective cell by cell separation and isolation.

 

Regardless of what method is chosen, once single cells are isolated, they are lysed in a manner that preserves cellular mRNA. The mRNA then helps to create complementary DNA (cDNA), which can be further amplified by polymerase chain reaction (PCR). After a sufficient amount of cDNA is amplified, sequencing libraries can be prepared. These sequencing libraries are powerful tools in downstream bioinformatic technologies and are particularly useful in single cell omics to classify the gene expression landscape in cells of a heterogeneous population.

FAQs:	Tools:
How do I prepare an RNA sequencing library?	RNA Molecular Weight Calculator RNA Concentration Calculator

scRNA-seq Technologies

---

Microfluidics Platforms

Various advances in scRNA-seq technologies have allowed for higher throughput of experiments, increasing the number of cells that can be isolated at once. Microfluidic technologies, for example, have increasingly been used in the field of scRNA-seq to allow for extremely efficient, highly scalable, single cell analysis and cell capture. By nature, microfluidic devices require minute amounts of starting samples, which reduces the necessary amount of chemicals and reagents in experimentation, reducing cost. Microfluidic systems are also easily automatable and are inherently isolated, so contamination of cells or reagents is of less concern.

One technology includes microwell arrays, where cells and barcoded beads can be loaded into microwells by repeatedly flowing them over the array until all of them are captured by gravity. This technology is particularly useful in minimizing sample degradation, and offers easy analysis, and provides the ability to tune steps in the technique upon cell loading. Some microwell scRNA-seq platforms have incorporated the use of optical or fluorescence imaging which can help determine marker composition and the viability of cells at different steps of the assay.

Another microfluidic technology amenable to scRNA-seq includes droplet-based systems. These systems, too, only require extremely low volumes of samples to screen thousands to millions of cells in a short time. In research, microfluidic scRNA-seq assays have been used for the large-scale analysis of tissues and tumors, and also for the analysis of rare cell types in sufficiently heterogeneous biological samples.

Barcoding

More recently, in situ barcoding concepts have been adapted to scRNA-seq. in situ barcoding was initially devised for single cell ATAC sequencing and later for whole genome sequencing. In in situ barcoding, single cells are never individually isolated. Instead, cells are split into mini-pools and distributed into multiwell plates that contain unique barcodes in each well. Then mRNA is manipulated and specifically labeled, in situ, inside of each cell. This procedure can be repeated over and over, so unlike traditional isolation methods, the number of potentially labeled cells can be exponentially scaled through each round of barcoding.

Spatial Transcriptomics

Spatial transcriptomics has also recently been used to advance scRNA-seq technologies. Spatial transcriptome analysis provides information about the spatial location of cells within its native tissue landscape. Incorporating spatial transcriptomics has helped improve the understanding of factors that determine morphology, genotype, and the microenvironment of cells. In particular, scRNA-seq combined with spatial transcriptomics can be especially beneficial in developing a precise diagnosis or effective treatment strategies for personalized medicine.

Assaywise Letter:

RNA Detection

Product Ordering Information

---

References

---

A practical guide to single-cell RNA-sequencing for biomedical research and clinical applications
Single-cell RNA sequencing technologies and applications: A brief overview
Single-cell RNA sequencing technologies and bioinformatics pipelines
Understanding Single Cell Sequencing, How It Works and Its Applications
An introduction to spatial transcriptomics for biomedical research