The problem with cancer tissue is that its intratumoral heterogeneity and its complexity is extremely high as cells possess, depending on their location and function, different mutations, different mRNA expression and the highest intricacy in the protein pattern. Prior to genomic and proteomic analyses, it is therefore indispensable to identify the exact part of the tissue or even the exact cell. Laser-based microdissection is a tried and tested technique able to produce pure and well-defined cell material for further analysis with proteomic and genomic techniques. It sheds light on the heterogeneity of cancer or other complex diseases and enables the identification of biomarkers. This review aims to raise awareness for the reconsideration of laser-based microdissection and seeks to present current state-of-the-art combinations with omic techniques.
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Article Synopsis
- Normal and malignant B cells have unique immunoglobulin (Ig) genes that can act as markers due to their high diversity.
- A laser-based microdissection method is used to isolate single B cells from frozen tissue sections and amplify specific rearranged Ig genes through semi-nested PCR.
- Analyzing these genes allows researchers to determine the clonal relationships of B cells, their gene usage, and their differentiation stage based on mutation patterns.
Microdissection-An Essential Prerequisite for Spatial Cancer Omics.
Proteomics
September 2020
Department of Otorhinolaryngology, MALDI Imaging and Core Unit Proteome Analysis, DFG Core Unit Jena Biophotonic and Imaging Laboratory (JBIL), Jena University Hospital, Am Klinikum 1, Jena, 07747, Germany.
The problem with cancer tissue is that its intratumoral heterogeneity and its complexity is extremely high as cells possess, depending on their location and function, different mutations, different mRNA expression and the highest intricacy in the protein pattern. Prior to genomic and proteomic analyses, it is therefore indispensable to identify the exact part of the tissue or even the exact cell. Laser-based microdissection is a tried and tested technique able to produce pure and well-defined cell material for further analysis with proteomic and genomic techniques.
View Article and Find Full Text PDFChromosome Microdissection on Semi-Archived Material.
Cytometry A
December 2019
Jena University Hospital, Institute of Human Genetics, Friedrich Schiller University, Jena, Germany.
Glass needle-based chromosome microdissection (midi) is a standard approach developed in the 1980s and remains more frequently applied in testing than the comparable technique using laser-based platforms. As the amount of DNA extracted by this technique is minimal and often in the range of picograms, the isolated DNA must be further amplified prior to use; the isolated amplified product can be readily utilized in multiple molecular research and diagnostic investigation. DNA libraries created by midi are either chromosome- or chromosome-region-specific.
View Article and Find Full Text PDFLaser-Based Microdissection of Single Cells from Tissue Sections and PCR Analysis of Rearranged Immunoglobulin Genes from Isolated Normal and Malignant Human B Cells.
Methods Mol Biol
July 2019
Senckenberg Institute of Pathology, University of Frankfurt, Frankfurt am Main, Germany.
Normal and malignant B cells carry rearranged immunoglobulin (Ig) variable region genes, which due to their practically limitless diversity represent ideal clonal markers for these cells. We describe here an approach to isolate single cells from frozen tissue sections by microdissection using a laser-based method. From the isolated cells, rearranged IgH and Igκ genes are amplified in a semi-nested PCR approach, using a collection of V gene subgroup-specific primers recognizing nearly all V genes together with primers for the J genes.
View Article and Find Full Text PDFHypoxia Sensing and Persistence Genes Are Expressed during the Intragranulomatous Survival of Mycobacterium tuberculosis.
Am J Respir Cell Mol Biol
May 2017
1 Tulane National Primate Research Center, Covington, Louisiana.
Although it is accepted that the environment within the granuloma profoundly affects Mycobacterium tuberculosis (Mtb) and infection outcome, our ability to understand Mtb gene expression in these niches has been limited. We determined intragranulomatous gene expression in human-like lung lesions derived from nonhuman primates with both active tuberculosis (ATB) and latent TB infection (LTBI). We employed a non-laser-based approach to microdissect individual lung lesions and interrogate the global transcriptome of Mtb within granulomas.
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