As a powerful genomics tool, RNA-Seq has a wide range of applications and can provide insights for research in biology and medicine. Whether in basic research, clinical diagnosis, tumor therapy, drug development, or personalized treatment, RNA-seq is playing an increasingly important role. As the technology continues to advance and the cost continues to decrease, the application scope of RNA-seq will be further expanded to help us better understand biological phenomena and provide stronger support for precision medicine and personalized treatment.
The following are some of the applications of RNA-seq:
RNA-Seq in Disease Research
RNA sequencing is revolutionizing disease research by offering a deep insight into gene expression patterns that can uncover potential biomarkers and therapeutic targets for various cancers.
Breast cancer is a type of cancer with a high prevalence among women in recent years, but there is no better treatment yet. To investigate the pathogenesis of breast cancer and to develop new therapeutic targets, the researchers obtained cancer cells from breast cancer patients and normal cells in the vicinity of the cancerous tissues, and studied the DEGs in the tumor tissues and healthy tissues by RNA-seq.
circCDYL was shown to promote the proliferation of breast cancer cells by enhancing autophagy gene expression and increasing autophagic flux. The circRNA circCDYL is highly expressed in cancerous tissues and serum of breast cancer patients, and this high expression increases the tumor load of patients. Especially in the serum of patients with more severe breast cancer disease, the expression of circCDYL is more abundant than in early-stage patients and patients with benign tumors in vivo, which leads to a lower survival rate in patients with severe breast cancer. And it was found that the more patients presenting low levels of circCDYL during chemotherapy, the higher the drug sensitivity of their clinical treatment, which suggests that high levels of circCDYL reduce the clinical outcome of patients. Meanwhile, the researchers identified a new circRNA by RNA-seq: CircSEPT9, which is formed by reverse splicing of exon 2 of the SEPT9 gene, and is a target of various transcription factors E2F1 and EIF4A3. Clinicopathological studies demonstrated a positive trend of circSEPT9 expression with T, N, and TNM staging, as well as a positive correlation with other clinicopathological parameters. circSEPT9 high expression in tumor tissues promotes the growth and metastasis of Orthotopic Xenograft. In conclusion, the researchers identified circCDYL and circSEPT9 by RNA-seq, both of which can be used as predictive factors to determine the extent of disease and clinical prognosis of breast cancer patients(Nicot C., 2020).
Bladder cancer (BC) is one of the most common malignant tumors of the urinary tract and one of the cancers with the highest mortality rate. However, no effective treatment has been found, and scientists have been working to find effective gene targets for BC. It has been widely reported in the literature that the Livin gene is highly expressed in bladder cancer patients and is able to influence the proliferation of cancer cells. The researchers used RNA-seq and qPCR to compare human bladder cancer cells with the human bladder cancer cell line T24, in which the Livin gene was knocked out, to identify the DEGs and to confirm the role of the Livin gene. More than 400 clinical RNA-seq samples (19 normal samples and 413 BC samples) were obtained and compared with the data measured by the researchers, and it was found that compared with normal cancer tissues, bladder cancer tissues knocked out of the Livin gene had low expression of the Livin gene and the proliferation, invasion and metastasis of the cancer cells were significantly reduced, whereas apoptosis was enhanced, which suggests that the Livin gene may play a role in bladder cancer disease. RNA-seq results showed that knockdown of Livin gene resulted in the up- or down-regulation of many cancer-related genes in tumor tissues, thus affecting various cancer-related pathways such as cancer cell proliferation and metastasis (Li X et al., 2023).
RNA-Seq in Drug Evaluation
At present, personalized medicine has become a major field of medical development, and personalized treatment has gone to achieve better therapeutic effects in the clinic, however, tumor heterogeneity has become a problem that hinders the progress of personalized medicine. While traditional bulk RNA-Seq can provide only average data of heterogeneous tumor populations, scRNA-seq allows researchers to examine the drug sensitivity and resistance of individual tumor cells. More RNA-seq techniques and their uses can be found in the following pages: "Overview of RNA Sequencing Techniques".
In a recent study, scRNA-seq was used to evaluate the sensitivity of various cancer cell lines to seven different drugs, revealing distinct responses across solid tumors and hematopoietic cells.
The drug sensitivity of seven drugs for different tumors was evaluated by migration learning from scRNA-seq profiles and drug response experimental datasets of three cell lines of different origins, showing that different solid tumors and hematopoietic cell lines respond to different drugs. Three drugs, gefitinib, afatinib and cetuximab, could target the EGFR signaling pathway, NVP-TAE684, sorafenib targeting the RTK signaling pathway, Vorinostat, and AR-42 targeting chromatin histone acetylation. And tumor biomarkers were inferred based on scRNA-seq (drug cetuximab as an example), which were compared with Buk RNA-seq, which were highly potentially useful, and three new biomarkers, TUBA1C, EIF4EBP1, and AHSA1, were identified.For the assessment of resistance, the combination of gefitinib and vorinostat combination could counteract resistance in most tumor cell populations, and it remains to be investigated whether other drugs can be used in combination(Zheng Z et al.,2023).
Identification of gene biomarker attributions to drug (Cetuximab) response heterogeneity(Zheng Z et al.,2023).
RNA-Seq in Gene Fusion and Mutation Detection
Gene fusion is a contributing factor to the pathogenesis of many diseases, especially cancer, and we need to accurately detect fusion genes in order to better diagnose the disease mechanism and develop better therapeutic approaches. RNA-Seq has been validated to be an effective tool to detect fusion genes, but there is a possibility of false positives.
To explore fusion genes in pediatric cancers, the researchers used RNA-seq and WGS to obtain transcriptomic and genomic data from children and developed Fusion-sq to detect fusion genes. In 128 pediatric pan-cancer in pediatric patients, the researchers detected 155 tumor-specific fusion genes and their structural variants and splicing modes, including fusion gene targets that have been therapeutically applied in the clinic, demonstrating that the Fusion-sq results were reliable and the targets identified were of high confidence. The researchers also identified 27 latent pathogenic fusion genes involving TSGs and oncogenes, and the discovery of these new fusion genes also provides new gene targets for the treatment and and prognosis of pediatric cancer (van Belzen IAEM et al., 2023).
RNA-Seq in Expression Profiling
RNA sequencing plays a crucial role in expression profiling by enabling the comprehensive analysis of gene expression patterns at the transcriptome level. This powerful technique helps researchers uncover genetic alterations in response to treatments, providing deeper insights into the underlying biological mechanisms.
5-aminolevulinic acid-mediated photodynamic therapy (5-ALA-PDT) is a new therapeutic approach to treating tumors, and there are no standardized criteria for what genetic changes occur during 5-ALA-PDT treatment. The researchers extracted primary secondary hyperparathyroidism (SHPT) cells from tumor patients and treated them, and used RNA-Seq to explore the genetic differences before and after the treatment, in order to analyze the therapeutic mechanism of 5-ALA-PDT. The RNA-Seq results identified more than 1,000 genes with up-regulation and 300 genes with down-regulation, and analyzed the function and pathway of these genes, and identified several genes. Pathway analysis identified several pathways such as TGF beta in the extracellular matrix (ECM), negative regulation of triglyceride biosynthesis process, and other processes, in addition to interactions between heat shock-associated proteins and key DEGs, and eight HSP genes with high probability of being involved in the therapeutic process of 5-ALA-PDT. In conclusion, the changes in expression profiles in SHPT progenitor cells during laser treatment were identified by RNA-Seq, providing us with further possibilities to analyze the mechanism of 5-ALA-PDT (Wen Y et al., 2023).
RNA-Seq in Plant and Animal Growth and Improvement
RNA-Seq has become a vital tool in understanding the genetic mechanisms underlying plant and animal growth and improvement. By providing detailed insights into gene expression across different developmental stages or environmental conditions, RNA-Seq enables the identification of key genes that control agronomic traits or physiological responses.
Cotton is an important cash crop in the world with high demand, and it is important for us to study the high yield of cotton. The formation and development of cotton fiber is important for the quality of cotton, especially cotton fiber is unicellular, which facilitates us to study the related genes that regulate the development in cotton fiber. The researchers isolated and characterized by scRNA-seq 14,535 cells in the cotton ovule periderm of the Xu142_LF line, which were at four developmental stages (1.5 days before anthesis, 1 day before anthesis, 0.5 days before anthesis, and on the day of anthesis), and determined these cells to establish the developmental process of early cotton fibers. Single cells were isolated by scRNA-seq and clusters of fibroblasts were established to identify specifically expressed genes into three categories: fibroblasts, non-fibroblastic epidermis and outer pigment layer. Exploration of fibroblast differentiation revealed that the early development of fibers proceeds roughly as precursor fibroblasts that have not yet begun to differentiate at -1.5 DPA, which differentiate into fibroblasts at -1 DPA and shift to diffuse growth fibroblasts that begin to start to protrude at -0.5 DPA, followed by a shift to tip-biased diffuse growth at 0 DPA. Analysis of the DEGs in the four processes identified a complex and sophisticated network of gene coordination and characterized the expression patterns of MYB25-like, HD1 and HOX3, three genes that play important roles in cotton fiber development, in the four processes (Qin Y et al., 2022).
Plant height, plant angle, plant height, thousand grain weight and so on are important agronomic traits in kale-type oilseed rape, and plant height has an important effect on yield in unfavorable environments such as big time. Plant height traits in oilseed rape are controlled by multiple genes, such as hormonal and non-hormonal pathways, and are strongly influenced by the environment. In order to explore the potential role of plant height improvement gene loci, the researchers conducted a combined analysis using BSA-seq and RNA-seq. The researchers chose two parents with different plant heights for analysis, and explored the key genes that led to the differences in plant height traits between the two parents. RNA-seq was performed on the stem tip and the upper part of the stem at the flowering stage, and the data identified genes that were significantly up-regulated and down-regulated, and combined with GO and KEGG analyses, the genes spanning the range between 63,989,634 and 64,945 bp on the chromosome of Brassica napus C02 and 64,945,122 bp on the C02 chromosome of Brassica napus were identified. ,122 bp spanning 955 kb between qPH_C02, a new plant height QTL, and identified eight possible candidate genes for PH improvement. The researchers also showed that a series of oxidative phosphorylation reactions carried out by the peroxisome may also affect plant height in oilseed rape (Xia J et al., 2024).
Studies have shown that brown adipose tissue (BAT) plays an important role in the stress response of animals, such as low temperature, high-fat diet and other such stimuli, and is related to animal obesity, is the energy dissipation organization of the animal's body, that is, it can reduce the occurrence of obesity. Acetate can regulate appetite, homeostasis and improve obesity in animals, but the regulatory relationship between acetate and BAT is still unclear, especially in cold environments, the potential role of acetate and BAT is not yet known. To explore the mechanism of action, the researchers characterized the effect of acetate on BAT in the cold environment by scRNA-seq. Both acetate and cold may activate BAT, and the potential association between the two is that pretreatment of acetate with drinking water followed by systemic administration may further enhance the activation of BAT in the cold environment, which suggests that systemic administration of acetate may promote the activation of BAT. However, the results also suggest that the results of systemic administration of acetate may be opposite to direct administration of acetate to specific tissues or organs, and that even the direct effects of acetate administration between brown adipocytes and brown adipose tissue are opposite. We also identified PPARs and C/EBPs, two regulatory genes that play important roles in the differentiation and development of BAT, which provide more clues for the analysis of the potential differentiation mechanisms (Ou H et al., 2023).
DEGs BAT samples from mice in the CE group and Ace_po+CE group (Ou H et al., 2023).
Virus identification
Plants encounter a variety of pathogenic fungi during their lifetime, but they have a unique set of methods to defend themselves against pathogenic fungi, i.e., PTI immunity. There are a series of genes specifically expressed in plant immunity, and in order to isolate and characterize these genes, there have been many reports in the literature to reveal the key DEGs in the immunity process by sequencing techniques such as RNA-seq, etc.
However, due to the fact that the reports were conducted independently, and due to the constraints of the environment and technology, there was no association of the data with each other. The researchers used published public RNA-seq databases (25 RNA-seq data from C. higginsianum-A. thaliana and B. cinerea-A. thaliana pathosystems) to jointly compare and analyze the core genes of Arabidopsis defense against two pathogens, Staphylococcus griseus and Cryptosporidium higginsianum. To ensure that the data were accurate and usable, the researchers performed a series of data normalization and data filtering, followed by WGCNA and DAVID analyses to explore the core genes. In the end, the researchers isolated and characterized roughly five core gene clusters responsive to a variety of pathogenic fungi, which suggests that RNA-seq databases have an important role to play in the improvement of our plant varieties (Soto-Cardinault C et al., 2023).
Framework for integrating publicly available RNA-seq data (Soto-Cardinault C et al., 2023).
Bovine viral diarrhea virus (BVDV) is a highly infectious virus in cattle farming because BVDV can be transmitted continuously, it has a defense strategy of "I run when the enemy comes, I infect the host when the enemy leaves," and it is continuously transmitted, and BVDV can evade host innate immunity. To further understand the mechanisms by which BVDV evades host innate immunity and persists in infection, the researchers explored key DEGs in bovine cells at different times during BVDV infection versus controls by RNA-Seq analysis. more than 1,000 DEGs were identified in infected MDBK cells at different times of the infection (2h, 6h, 12h, and 24h), and the study showed that BVDV infection induced the up-regulation of genes involved in lipid metabolism in host cells, while the expression of genes with antiviral effects (including ISG15, Mx1, and OSA1Y) were down-regulated, which may also contribute to the ability of BVDV to evade the innate immune mechanism of the host. The expression of some genes related to the host complement system, such as C1R, SERPINE1, KNG1 and C1S, was also down-regulated, which may indicate that BVDV inhibits the expression of antiviral proteins in the host complement system and thus promotes its proliferation (Liu C et al., 2019).
Selective Splicing
Eukaryotic genes need to be spliced and processed in order to be transformed into mature mRNAs, and selective splicing is one of the common mechanisms in eukaryotes. Moreover, studies in various organisms have shown that selective splicing is closely related to the occurrence of diseases, and AS can also affect the cellular localization of genes or proteins, so we need to understand the AS events of genes in order to better understand the specific expression of a gene. To investigate how variable splicing affects the subcellular localization of RNA the researchers performed RNA-seq, and by analyzing RNA-seq histology data from the cytoplasm and nucleus of 13 human cell lines, it was observed that variable splicing affects the localization of RNAs in both the cytoplasm and nucleus as well as the expression imbalance. Some variably spliced forms of RNA that retained introns preferred to localize in the nucleus. Analysis of the introns in the transcripts of these variable spliced transcripts that retained introns revealed that they contained 14 RBPs that bind readily to these short and stable introns, and nuclear localization signals were known to be present on some of these RBPs based on previous reports (Zeng C et al., 2020).
Rainbow trout (Oncorhynchus mykiss) is one of the cold-water cultured economic fishes, which is sensitive to heat stress. It has been shown that selective splicing (AS) is an important regulatory mechanism for the expression of genes into proteins in cells. In order to investigate whether selective splicing plays a role in the response to heat stress in Oncorhynchus mykiss, the researchers identified more than 18,600 selective splicing events from approximately 10,000 genes and 2,100 DEGs using RNA-Seq. The researchers used RNA-Seq to identify more than 18,600 selective splicing events from approximately 10,000 genes and 2,700 differential selective splicing (DAS) events from 2,100 DEGs. Functional and pathway analyses of these DEGs revealed that they are involved in cellular heat stress and cellular metabolic responses, especially the lysine degradation pathway and other pathways involved in the response of Oncorhynchus mykiss liver tissue to heat stress (Sun J et al., 2022).
References:
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- Li X, Fu C, Li G, He H."RNA-seq reveals novel mechanistic targets of Livin in bladder cancer." BMC Urol. 2023 Feb 28;23(1):26.doi: 10.1186/s12894-023-01194-w
- Zheng Z, Chen J, Chen X, Huang L, Xie W, Lin Q, Li X, Wong KC."Enabling Single-Cell Drug Response Annotations from Bulk RNA-Seq Using SCAD."Adv Sci (Weinh).2023;10(11):e2204113.doi: 10.1002/advs.202204113
- van Belzen IAEM, Cai C, van Tuil M, Badloe S, Strengman E, Janse A, Verwiel ETP, van der Leest DFM, Kester L, Molenaar JJ, Meijerink J, Drost J, Peng WC, Kerstens HHD, Tops BBJ, Holstege FCP, Kemmeren P, Hehir-Kwa JY."Systematic discovery of gene fusions in pediatric cancer by integrating RNA-seq and WGS." BMC Cancer. 2023 Jul 3;23(1):618.doi: 10.1186/s12885-023-11054-3
- Wen Y, Zeng L, Chen Q, Li Y, Fu M, Wang Z, Liu H, Li X, Huang P, Wu W, Zou Q, Yi W. "RNA-Seq-based transcriptomics analysis during the photodynamic therapy of primary cells in secondary hyperparathyroidism." Photochem Photobiol Sci. 2023 Apr;22(4):905-917.doi:10.1007/s43630-023-00361-0
- Qin Y, Sun M, Li W, Xu M, Shao L, Liu Y, Zhao G, Liu Z, Xu Z, You J, Ye Z, Xu J, Yang X, Wang M, Lindsey K, Zhang X, Tu L."Single-cell RNA-seq reveals fate determination control of an individual fibre cell initiation in cotton (Gossypium hirsutum)."Plant Biotechnol J. 2022 Dec;20(12):2372-2388.doi: 10.1111/pbi.13918
- Xia J, Zhan L, Zhang J, Song W, Xu X."Identification of Rapeseed (Brassica napus L.) Plant Height-Associated QTL Using BSA-seq and RNA-seq." Int J Mol Sci. 2024 Sep 12;25(18):9875.doi: 10.3390/ijms25189875
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- Ou H, Chen Q, Lin Z, Yang Y, Wang P, Sriboonvorakul N, Lin S."RNA-seq Analysis Reveals Potential Synergic Effects of Acetate and Cold Exposure on Interscapular Brown Adipose Tissue in Mice." Biology (Basel). 2023 Sep 26;12(10):1285.doi: 10.3390/biology12101285
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- Sun J, Liu Z, Quan J, Li L, Zhao G, Lu J."RNA-seq Analysis Reveals Alternative Splicing Under Heat Stress in Rainbow Trout (Oncorhynchus mykiss)." Mar Biotechnol (NY). 2022 Mar;24(1):5-17.doi: 10.1007/s10126-021-10082-z
