The Booming RNA Sequencing Technologies in Cancer Research

RNA sequencing (RNA-Seq) is a common approach in the life sciences that has been used extensively in cancer research, cancer diagnosis and prognosis, and drug development. RNA-Seq techniques have rapidly advanced from bulk RNA-Seq, laser-captured micro-dissected RNA-Seq, and single-cell RNA-Seq to digital spatial RNA profiling, spatial transcriptomics, and direct in situ sequencing, powered by a variety of biological and technical questions. In the field of clinical oncology, each of these technologies has its own set of strengths, weaknesses, and applications.

RNA Sequencing Technologies and Their Applications

Figure 1. Applications of RNA-seq in differential expression analysis and cancer biomarkers, cancer heterogeneity and drug resistance, cancer immune microenvironment, immunotherapy and neoantigen. (Hong, 2020)

Bulk RNA-Seq

Bulk RNA-Seq is the most common transcriptomic technique for examining the transcriptional landscape and modified molecular pathways in human cancers. Total RNA extraction, library construction, sequencing, and data analysis are the four key steps in RNA-Seq. The form of the library used, kit selection, sequencing type, and sequencing depth will all be determined by the biological question and RNA quality.

For both cancer research and clinical uses, bulk RNA-Seq is a cost-effective and effective tool. Clinical RNA-Seq is now primarily used in the exploration of novel gene fusions, panel-based gene fusion diagnosis, whole transcriptome-based biomarker discovery, and therapeutic treatment guidelines.

Laser Capture Micro-Dissected RNA-Seq

Laser capture microdissected RNA-Seq is one of the most straightforward methods (LCM-RNA-Seq). Laser capture microdissection of cells of interest, accompanied by normal RNA-Seq, are the key processes in LCM-RNA-Seq. Although FFPE materials are used in the majority of LCM-RNA-Seq experiments, the RNA extracted from them is notoriously low in quantity and quality, and the LCM processes further decrease RNA integrity.

Single-Cell RNA-Seq

For single-cell RNA-Seq, there are a variety of innovations currently available (scRNA-Seq). One of the first scRNA-Seq technologies was the Fluidigm C1 microfluidics system. In a single day, this system can only process 96 single cells. Using enhanced Fluidigm IFC chips, the throughput is improved to 800 single cells. Microdroplet-based single-cell sequencing systems have recently risen to prominence. Unlike LCM-RNA-Seq, Chromium technology allows for the rapid analysis of gene expression profiles of up to 10,000 individual cells in a single experiment.

Digital Spatial Profiling

Digital spatial profiling (DSP) technology, which was recently developed, has made it easier to fix spatial gene expression with substantially increased throughput. The assay depends on RNA hybridization probes conjugated to photocleavable oligonucleotide tags as its main technology. The slide is imaged after probes attach to their directed mRNA on the slide-mounted FFPE tissue sections. After that, UV exposure is used to release the oligonucleotide tags from the tissue's regions of interest. The number of tags that have been released is counted. The counts of a specific tag, which represents a specific mRNA, are mapped back to tissue location (defined region of interest) to produce a spatially resolved digital profile of mRNA abundance.

Spatial Transcriptomics

For several years, researchers have been working on a new spatial transcriptome technology. By allowing scientists to study the entire transcriptome spatially, this technology overcomes the limitations of DSP technology. It has the potential to provide data similar to bulk transcriptome analysis, as well as spatial content. Fresh-frozen mammalian tissues and fresh plant tissues, as well as FFPE materials, can be utilized for spatial transcriptomic assessment. This powerful technology's application in the cancer arena is still limited, and it's only being tested in early technology access.

Fourth-Generation RNA-Seq

The ultimate objective of RNA-Seq is a single-cell resolution, simple, robust, spatially resolved transcriptomic analysis. Fourth-generation sequencing technologies such as in situ sequencing (ISS) and fluorescent ISS (FISSEQ), which have recently been developed, have the potential to lead to this destination.

References:

1. Hong M, Tao S, Zhang L, et al. RNA sequencing: new technologies and applications in cancer research. Journal of hematology & oncology. 2020 Dec;13(1).
2. Wang J, Dean DC, Hornicek FJ, et al. RNA sequencing (RNA-Seq) and its application in ovarian cancer. Gynecologic oncology. 2019 Jan 1;152(1).
3. Zhu S, Qing T, Zheng Y, et al. Advances in single-cell RNA sequencing and its applications in cancer research. Oncotarget. 2017 Aug 8;8(32).