Sequencing technologies have expanded the possibilities to a single-cell – and even subcellular- resolution. The ability to read the sequence from intact tissues is critical to understand complex biological and pathological mechanisms. In situ sequencing offers an in-depth understanding of gene expression and its location. 

What is In Situ Sequencing? 

In situ sequencing (ISS) is a targeted and barcoded method where RNA is sequenced directly in a morphologically preserved tissue or cell sample. Unlike traditional sequencing methods, where samples are analyzed after extraction -where histological context is lost, ISS simultaneously analyses hundreds of mRNA transcripts and their spatial context at cellular and subcellular resolution. Developed by Mats Nilsson’s group at the Science for Life Laboratory at Stockholm University, ISS allows scientists to identify cell types by analyzing their gene expression profiles. 

In Situ Sequencing Benefits 

In situ sequencing is becoming the method of choice for scientists studying gene expression, pathological mechanisms, and new drug targets. 

• High efficiency in detecting multiple RNA molecules in a tissue section in a high-throughput manner, covering a comprehensive dynamic range 
• Provides spatial information of gene expression profiles for gene panels at subcellular resolution 
• Capability to include analysis of multiple genes across various tissue types 
• Ability to identify point mutations in preserved cells and tissue 
• High specificity for targeting single nucleotide variations within tissue, which makes it a helpful tool in pinpointing somatic mutations 

In Situ Sequencing Applications 

In Situ Sequencing as a Diagnostic Tool in Cancer 

With ISS capabilities of multiplexing gene expression and the ability to identify point mutations in fixed cells and tissue, it is a powerful diagnostic tool for cancer tumors. The first such application was presented in the early 2013 study by Mats Nelisson when ISS was used across human breast cancer tissue. So far, ISS has been used to analyze many more genes across various tissues. 

In Situ Sequencing Integration with Other Tools 

ISS can be incorporated with other methods like single-cell RNA sequencing and Spatial Transcriptomics, which can be valuable for cell mapping and analysis of the cellular network. For example, a recent study used spatial transcriptomics and in situ sequencing to demonstrate the role of amyloid-beta plaques linked to Alzheimer’s disease. 

In Situ Sequencing Protocol 

In situ sequencing is a targeted method using padlock probes (PLPs), rolling circle amplification (RCA) and sequence by ligation. These are the primary steps of the current protocol: 

• mRNA generates cDNA by reverse transcription 
• PLP hybridization: A padlock probe hybridization to the targeted cDNA fragment 
• DNA ligation closes the two probe ends to form a circularized DNA 
• PLPs are amplified by targeted RCA, which generates Rolling Circle Products (RCPs) 
• RCPs are sequenced by ligation 
• The sample is read using imaging technologies, and each RCP reveals a color to the corresponding base 
• The process is repeated to achieve the number of preferred reads of bases. 

Design Your In Situ Sequencing Experiment 

At OmicsVeu, we developed NanoVIP^®, an automated In Situ Sequencing solution. See the video to learn more about NanoVIP^®.

Conclusion 

In situ sequencing is a targeted image-based technology that can detect gene expression and provide location information within a cell sample or a tissue section. This tool is valuable for understanding the pathology and molecular mechanisms of diseases. So far, it has been applied in various areas, such as cancer diagnosis, gene expression profiling, and mutation detection. Providing gene expression images and spatial context using ISS results in an atlas-like map which has been used to develop The Human Developmental Cell Atlas. The project is a Swedish initiative developed by the Science for Life Laboratory, which aims to create a detailed molecular atlas of human cell types and show their spatial distributions in three dimensions. 

References: 

• Chen, W.-T. et al. (2020) “Spatial transcriptomics and in situ sequencing to study alzheimer’s disease,” Cell, 182(4). Available at: https://doi.org/10.1016/j.cell.2020.06.038. 
• Hilscher, M.M. et al. (2021) “Correction to: In situ sequencing: A high-throughput, multi-targeted gene expression profiling technique for cell typing in tissue sections,” Methods in Molecular Biology [Preprint]. Available at: https://doi.org/10.1007/978-1-0716-0623-0_25. 
• In situ sequencing (2022) SciLifeLab. Available at: https://www.scilifelab.se/units/in-situ-sequencing/ (Accessed: December 4, 2022). 
• Ke, R., Mignardi, M., Pacureanu, A. et al. In situ sequencing for RNA analysis in preserved tissue and cells. Nat Methods 10, 857–860 (2013). https://doi.org/10.1038/nmeth.2563 
• The Human Developmental Cell Atlas (no date) HDCA Sweden. Available at: https://hdca-sweden.scilifelab.se/ (Accessed: December 4, 2022).