Spatial Biology – Made Easy

Spatial biology is a field that studies the spatial distribution and interaction of biomolecules, viz., DNA, RNA, proteins, and metabolites in their native tissue context. Spatial Multiplex Multiomics is a field of spatial biology that aims to measure and map these multiple types of biomolecules (DNA, RNA, proteins, and metabolites) in native tissue context which can reveal the complex interactions and functions of cells and tissues in health and disease.

One of the leading providers of spatial multiplex multiomics products and services is Omicsveu, a company that offers a range of technologies, such as Phenoveu, RNAveu, miRNAveu, and In Situ Sequencing that enable the detection and visualization of multiple biomarkers in the same tissue section.  

Phenoveu: Allows simultaneous detection of multiple protein biomarkers using the same tissue section. 

RNAveu: Allows the detection of mRNA expression in sample with sub-micron level accuracy by using uniquely labelled fluorophore-tagged barcoded probes. 

miRNAveu: Allows the detection of target miRNA expression and can be combined with other technologies such as RNAveu and Phenoveu. 

In situ Sequencing technology: Examine the location of target mRNA sequences along with changes in single base pair mutations at the tissue and single cell level. 

RNAveu and Phenoveu

Omicsveu also provides a fully automated staining system, NanoVIP, that can process up to 64 In Situ spatial reactions or up to 96 sequencing-ready libraries in a ~2 days process. NanoVIP system offers a broad line of Spatial Multiplex Multiomics products consisting of a fully automated staining system, ready-to-use probes, multiplexing antibodies and visualization kits. Omicsveu’s technologies have been used to study various biological processes and diseases, such as tumor development and progression, immune response, and organ development. 

Some of our recent publications: 

 

 

 

 

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Advancements in Multiomics: MicroRNAs in Tumor Development and Progression using miRveu Technology Developed by Omicsveu

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Introduction

MicroRNAs (miRNAs) are endogenous, non-coding RNAs known to regulate gene expression by translational repression or RNA cleavage and play important role in carcinogenesis and metastasis. Due to their well-established selectivity and specificity, miRNAs can represent a useful tool, both in diagnosis and therapy, in forging the path towards the achievement of precision medicine. At OmicsVeu, we have developed Super SensitiveTM microRNA probes that can be used to identify miRNA expression profile in different grades of cancer, with high sensitivity and specificity. This blog peeks into the world of miRNAs followed by its role in carcinogenesis, development of miRNA probes, their applications using NanoVIP®.

MiRNAs and their role in carcinogenesis

miRNAs regulate transcription by binding to complementary sequences in the target mRNA, usually in the 3’ untranslated region, leading to mRNA degradation or translational repression, thereby influencing protein expression levels. miRNAs have been implicated in a wide range of biological processes such as cell differentiation, proliferation, apoptosis, organogenesis, morphogenesis, immune response, and disease pathogenesis.

 

Utilizing miRNAs as a potential biomarker to differentiate tumors will allow for specialized treatment regimens and increased clinical care. Numerous studies have demonstrated that aberrantly expressed miRNAs are involved in the pathogenesis of the aggressive triple negative breast cancer (TNBC) phenotype. BRCA mutated breast carcinomas are commonly seen in younger patients and have a more aggressive clinical course. Similarly, miRNA expression profiles were evaluated in a study in formalin-fixed paraffin-embedded (FFPE) cases of different grades of prostate cancer, including paired normal prostate, benign prostatic hyperplasia (BPH) and high-grade prostate cancer. miR-17 was deregulated in prostate cancer IV tissues and miR-125b was strongly upregulated in over 70% of cases of prostate cancer IV and III tissues. In benign prostatic hyperplasia (BPH) tissues, only miR-205 was significantly upregulated. The study findings underscore the importance of visualizing miRNA expression to differentiate cancer and benign cells with spatial differentiation in FFPE tumor tissues.

Super SensitiveTM miRNA probes

Omicsveu has developed proprietary Super SensitiveTM miRNA probes that enhance signals from intrinsically low abundant miRNAs. These oligos are synthesized using specially designed bases to give high melting temperatures. The high melting temperature oligonucleotide probes enable stringent washes at elevated temperatures to remove non-specific binding. Our miRNA probes are dual end labelled on 3’ and 5’ with five fluorescent molecules that amplify the signal, giving high resolution stains. These probes enable detection of low expressed miRNAs thus allowing high sensitivity and specificity (Figure 1). Our miRNA probes are currently one of the best products for complex diagnostic assays. Over 220 ready-to-use miRNA probes are now available and more are being developed.Hybridization to target miRNA

Figure 1. Super SensitiveTM miRNA probe developed by Omicsveu

NanoVIP® for miRVeu

The high-density fluorophore-labelled miRNA probes combined with our fully automated staining system NanoVIP® aids in studying the low expressed miRNA populations and assess its expression profiles along with the physiological function.  NanoVIP® automated staining procedure is complete xylene-free system. It incorporates multiple rounds of staining, stripping, and removal of coverslip to prepare for second multiplex run. miRNA probes combined with the automated processing using NanoVIP® greatly increases the reliability of the test results.

Apart from the above, our miRVeu detection kits are optimized for miRNA detection with robust amplification of the signal producing clean intense staining. Each kit includes easy-to-follow protocols and ready-to-use reagents. Due to high sensitivity and specificity of the probes, these kits can be used for either manual staining or high-throughput, automated staining. Figure 2 illustrates results of miR-541 in pancreatic tumor tissue using miRveu technology in combination with NanoVIP®HSA-miR-541

Figure 2: Expresson of miR-541 on pancreatic tumor tissue detected through miRVeu technology using NanoVIP

miRveu Potential Applications

The miRNA expression profiles are used to assess:

  • Classification of cancer subtypes – Due to their well-established selectivity and specificity, miRNAs can represent a useful tool, both in diagnosis and therapy, in forging the path towards the achievement of precision medicine.
  • Undifferentiated and poorly differentiated tumors – miRNAs are multifaceted and can be used for differentiation of malignant and benign tumors, early-stage cancer detection marker, identification of cancer of unknown primary, and classifying undifferentiated and poorly differentiated tumors.

Grading and staging of cancer – miRNA expression profiles can be used to unambiguously identify miRNA expression profile in different grades of cancer, with high sensitivity and specificity. It also helps in staging and differentiation of cancer subtypes.

Conclusion

miRNAs have emerged as remarkable players in the complex world of carcinogenesis, demonstrating their pivotal roles in regulating gene expression and cellular processes that can ultimately lead to cancer development. Their ability to serve as biomarkers for early cancer detection, as well as prognostic indicators for disease progression, holds great promise in improving patient outcomes. NanoVIP® automated systems, miRNA Probes and miRveu technology can be used to successfully differentiate miRNA expression patterns and implement its diagnostic and therapeutic properties. While there is still much to discover, the journey into the world of miRNAs and their applications in cancer research and therapy is one filled with hope and potential breakthroughs.

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In Situ Sequencing: Benefits, Applications, and Protocol

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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 Methods10, 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).

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ImmunoPlex, A Sequential Immunofluorescence System Provides Insights Into The Tumor Microenvironment

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Infographic Transcript

ImmunoPlex, a sequential immunofluorescence system provides insights into the tumor microenvironment

ImmunoPlex assay, a multiplex immunofluorescence system provides simultaneous detection of multiple protein biomarkers in a single tissue section. OmicsVeu offers ImmunoPlex DNA Barcode antibodies along with optimized ready-to-use kits for the simultaneous detection of 40 to 60 biomarkers in single tissue sections.

OmicsVeu’s ImmunoPlex is designed for translational researchers to reveal high-end protein signature profiles while preserving morphology with a low turnaround time for immuno-oncology applications.

  • OmicsVeu offers high-temperature stripping technology, preservers morphology, and supports up to 60 iterations on the same slide.
  • Our wide range of oligonucleotide-conjugated antibodies with the fluorophore-conjugated complementary strand gives intense, crisp, reliable, and consistent results.
  • Our system produces results with more than 4 fluorophores in every run.
  • NanoVIP® automates ImmunoPlex protocol in a wide range of samples including FFPE tissues with minimum hands-on time.

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