How To Validate Single-Cell RNA-Seq Data?
InquirySingle-cell RNA sequencing (scRNA-seq) is a powerful technology enabling high-throughput sequencing and transcriptome analysis at the individual cell level. The significance of validating scRNA-seq data lies in guaranteeing the precision and dependability of results derived from this genomic approach. Beyond merely mapping key cell types and identifying differential genes, contemporary single-cell research transcends these boundaries and demands downstream validation experiments for a more comprehensive research framework and credible conclusions.
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What Are the Techniques for Spatial Transcriptomics?
Validation plays a pivotal role in establishing the trustworthiness of scRNA-seq data in complex biological systems. This involves executing quality control measures, confirming biological relevance, addressing normalization and batch effects, accurately identifying cell types, bolstering statistical rigor, ensuring result replication and robustness, tackling data integration challenges, and aligning with publication standards. Various validation methods are elucidated in the subsequent discussion, underlining their essential contribution to the reliability of scRNA-seq findings.
CD Genomics excels in spatial omics and single-cell sequencing technology, providing top-notch solutions for life sciences. We provide single-cell transcriptome sequencing services based on the 10x Genomics and Smart-Seq2 technology platforms, covering material selection, library construction, and sequencing to data analysis. Meanwhile, every step has been carefully designed and scientifically optimized to ensure high-quality research results.
Using RNA FISH to Validate Single-Cell RNA-Seq Data
The validation of single-cell data through RNA Fluorescence in situ Hybridization (RNA FISH) is a technique employing fluorescently labeled exogenous nucleic acid fragments as probes. These probes, complementary to the RNA of interest, form specific nucleic acid hybrid molecules when applied to tissues, cells, or chromosomes, revealing the precise spatial location of the targeted RNA in a fluorescence detection system. RNA FISH serves as a prevalent validation method in single-cell research, not only facilitating the detection of marker gene expression levels but also enabling the determination of the spatial localization of a marker gene-labeled cell population within tissues. This method proves particularly valuable in discerning the predominant cellular states in tumors, such as mesenchymal-like (MES) or progenitor-like (PROG) cells. RNA FISH assays contribute to validating the presence and distribution of these distinct cellular states within individual tumors, enhancing our understanding of tumor heterogeneity.
IF for Validation of Single-Cell RNA-Seq Data
The Immunofluorescence technique (IF) operates on the principle of the specific binding between antigens and antibodies. In this method, a fluorescent pigment, non-interfering with the antigen-antibody activity, is labeled onto the antibody (or antigen). This labeled antibody then binds to its corresponding antigen (or antibody) being tested, exhibiting a distinct fluorescent reaction under a fluorescence microscope. IF serves as a protein-level validation experiment, aiming to qualitatively characterize the expression levels and spatial localization of marker gene proteins in tissues. A study utilized multiple immunofluorescence assays to further validate the presence of tumor-associated natural killer cells (TaNK cells) in cancer, providing crucial insights into the cellular dynamics within the tumor microenvironment.
IHC and Single-Cell Transcriptome Analysis
Immunohistochemistry (IHC) operates on the principle of specific antigen-antibody binding, utilizing antibodies to label proteins within tissue cells. The location and expression levels of these proteins within the tissue cells are then determined through a chemical reaction that induces the development of color in the antibodies. IHC, akin to Immunofluorescence (IF), serves as a protein-level validation assay.
Researchers validated the significant correlation of NPTX2 expression with cognitive function. They demonstrated a noteworthy reduction in NPTX2 expression in older cognitively impaired individuals using IHC, aligning with the findings from single-cell transcriptome analyses. This integration of IHC results reinforces the association between NPTX2 and cognitive function in the context of aging and cognitive impairment.
Overlapping fraction of genes positively associated at the different stages of disease progression in the PFC with genes positively associated at the different stages of disease progression in the MTG. (Mathys et al., 2023)
Specific Cell Sorting Accelerating Single-Cell Transcriptome Analysis
Specific cell population sorting involves the targeted selection of desired cells using flow or magnetic bead sorting, relying on cell membrane or intracellular markers post-transfection with fluorescent markers. This is followed by RT-qPCR to assess the expression of target genes. This technique, predominantly employed in single-cell studies, serves to validate cell subpopulation ratios or identify novel cell subpopulations.
In research, various immune cell types such as macrophages, neutrophils, NK cells, B cells, CD4+ cells, and CD8+ T cells were meticulously sorted using flow assay. The sorted populations were then compared with the results of scRNA-seq, demonstrating a consistent alignment between the two methods. This integration of specific cell population sorting enhances the reliability of single-cell analyses by corroborating and validating findings across different experimental approaches.
Gene Overexpression, Silencing, And Knockout Techniques
Gene overexpression, silencing, and knockout techniques serve as pivotal tools for validating the functionality of key marker genes identified through single-cell sequencing, offering deeper insights into the associated molecular mechanisms. These methods can be broadly categorized into function acquisition and function inactivation. Gene overexpression is employed for function acquisition, whereas techniques such as RNA interference and CRISPR/Cas9 knockdown are utilized for function inactivation.
41 hormone-responsive genes, including LAX2, LAX1, and LOX3, were discerned in cotton through scRNA-seq. The authors further substantiated the roles of these genes by constructing GhLAX1 and GhLOX3 knockout plants (CR1 and CR2, respectively) using CRISPR/Cas9 technology. Additionally, gene overexpression techniques were employed to create overexpression plants (OE1) of GhLAX2. These manipulations provided crucial evidence for the significant involvement of LAX2, LAX1, and LOX3 in the proliferation of healing tissues and plant regeneration.
Integrating Single-Cell Transcriptomes with Single-Cell ATAC-Seq and Spatial Transcriptomes
Integrating single-cell transcriptomes with single-cell ATAC-seq and spatial transcriptomes for multi-omics research not only serves to validate and reinforce each other's findings but also offers a comprehensive understanding of biological processes.
CD Genomics excels in spatial omics and single-cell sequencing technology, providing top-notch solutions for life sciences. Using advanced tools like microscopy and spatial sequencing, we analyze genomics, transcriptomics, proteomics, and metabolomics in complex tissues with high resolution. Our goal is to create user-friendly spatial omics solutions and we are open to collaborations in various research fields to explore new areas together.
Using the article as an illustration, scRNA-seq was employed to identify ADAMTS4 in lung tissues of influenza A virus-infected mice. Notably, the expression of ADAMTS4 was confined to non-immune stromal cells and was significantly up-regulated in fibroblasts post-infection. To elucidate the spatial localization of ADAMTS4, spatial transcriptome analysis was conducted on lung tissues from mice experiencing severe respiratory distress (day 10 post-infection). This analysis revealed that ADAMTS4 expression was localized to interstitial inflammatory zones near the alveoli. The integration of single-cell techniques and spatial transcriptomics provided valuable insights, suggesting ADAMTS4 as a potential therapeutic target for intervention in cases of respiratory distress associated with influenza A virus infection.
Reference
- Mathys, Hansruedi, et al. "Single-cell atlas reveals correlates of high cognitive function, dementia, and resilience to Alzheimer's disease pathology." Cell 186.20 (2023): 4365-4385.
