Application of Laser Microdissection to Uncover Regional Transcriptomics in Human Kidney Tissue
June 9th, 2020
We describe a protocol for laser microdissection of sub-segments of the human kidney, including the glomerulus, proximal tubule, thick ascending limb, collecting duct and interstitium. The RNA is then isolated from the obtained compartments and RNA sequencing is carried out to determine changes in the transcriptomic signature within each sub-segment.
Laser microdissection allows for the transcriptomic analysis of spatially defined regions within the kidney tissue samples. This enables us the unique opportunity to identify structures of interest even after disease induced changes took place. The main advantages of this methodology are its complementarity to other omics technologies, such as single cell RNA sequencing.
It affords remarkable tissue economy and even allows the detection of lowly expressed transcripts. After acquiring the tissue sample, use a cryostat set to minus 20 degrees Celsius to cut the specimen to a thickness of 12 micrometers and use the slide adapter to fix the specimen to a specialized laser microdissection slide. Within 10 days of cryosectioning, mix 89.2 microliters of RNase-free 10%BSA in PBS with four microliters of FITC phalloidin, 1.5 microliters of DAPI, two microliters of the antibody of interest directly conjugated to Alexa Fluor 546, and 3.3 microliters of RNase inhibitor.
Wash the slide in minus 20 degrees Celsius 100%acetone for one minute and place the slide in a humidity chamber. Wash the top of the slide two times with fresh RNase-free PBS for 30 seconds per wash, followed by two 30 second washes with 10%BSA in RNase-free PBS. After the last wash, treat the sample with the prepared antibody solution for five minutes at room temperature before washing two more times with 10%BSA in RNase-free PBS.
After the second wash, air dry the slide for five minutes in a tissue culture hood before loading the slide onto the laser microdissection cutting platform. Install autoclaved 500 microliter micro-centrifuge collection tubes, appropriate for PCR work, containing 50 microliters of extraction buffer from an RNA isolation kit onto the device. After loading, identify regions of interest within the sample by staining, morphology, and location.
Use immunofluorescence to outline at least 500, 000 micrometer square segments of interest. Then initiate the laser microdissection to excise the region using a laser power of greater than 40. Upon completion of the laser microdissection process, cap the microcentrifuge collection tubes and flick the tubes vigorously to ensure that the contents move from the caps to the bottoms of the tubes.
After centrifugation, incubate the tubes in a 42 degrees Celsius water bath for 30 minutes before centrifuging the tubes again. Then transfer the supernatants to new 500 microliter tubes from minus 80 degrees Celsius storage. The identification of tubular sub-segments in the kidney is accomplished through antibody staining of unique tubular markers in addition to cell morphology and structural landmarks.
Fluorescence labeling in conjunction with morphological tissue features and the spatial positioning of the image structures facilitates the visualization of renal sub-segments with a high degree of confidence. In this representative downstream sequencing analysis, the success rate of meeting the minimum dissection area input was greater than 90%resulting in a high total mRNA quantity available for gene detection. In this analysis, 100%of the samples met the minimum detection threshold of at least 10, 000 genes.
After laser microdissection, enrichment analysis of the gene expression of known markers can be compared to those from other compartments. In this analysis, one marker for each sub-segment was identified through laser microdissection regional transcriptomics that also yielded a specific immunohistochemical staining of the corresponding nephron sub-segment. It's important to note that laser microdissection relies heavily on the expertise of the user to precisely identify the structures of interest to be dissected.
This technique allows the study of specific regional gene expression signatures that can be used to assess potential pathways involved in disease progression, as well as the biology of healthy tissue.
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Overview
This study presents a protocol for laser microdissection of specific sub-segments within human kidney tissue, including the glomerulus and proximal tubule. The isolated RNA from these compartments enables transcriptomic analysis to reveal changes in gene expression patterns associated with different kidney structures.
Key Study Components
Research Area
- Transcriptomic analysis
- Kidney biology
- Disease progression investigation
Background
- Importance of studying kidney sub-segments
- Challenges in analyzing spatially defined structures post-disease
- Complementarity with other omics technologies
Methods Used
- Laser microdissection
- Human kidney tissue samples
- RNA isolation and sequencing
Main Results
- High success rate in dissection and RNA recovery
- Identification of gene expression markers for kidney sub-segments
- Potential pathways related to disease progression demonstrated
Conclusions
- This protocol demonstrates an effective method for studying regional gene expression in the kidney.
- Findings contribute to understanding renal biology and potential disease mechanisms.
Frequently Asked Questions
Executive Industry Relevance
Laser microdissection enables spatially resolved transcriptomic profiling of human kidney tissue, addressing a critical gap in discovery biology where dissociation-based methods lose anatomical context. This approach supports target validation by linking gene expression signatures to defined nephron sub-segments, enhancing mechanistic de-risking in renal disease models. The method provides predictive confidence for pathway interrogation and portfolio prioritization in early discovery workflows.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses by mapping gene expression to anatomically defined kidney compartments.
- Operational Value: Supports biological de-risking through spatially anchored transcriptomic signatures that maintain tissue context.
- Predictive Value: Facilitates target confidence by correlating regional marker expression with functional sub-segment identity.
Screening & Assay Development
- Scientific Value: Prepares validated biological systems for downstream omics workflows by isolating high-integrity RNA from microdissected regions.
- Operational Value: Ensures assay standardization and reproducibility through QC parameters assessing specimen adequacy and dissection success rates.
- Scalability: Enables reliable compound evaluation by providing quantitative, spatially resolved transcriptomic outputs for screening campaigns.
Translational & Preclinical Research
- Translational Continuity: Connects discovery findings to preclinical validation through disease-relevant system modeling using human kidney sub-segments.
- Biomarker Alignment: Supports translational biomarker development by identifying compartment-specific gene expression profiles detectable in tissue.
- Risk-Adjusted Advancement: Informs preclinical decision-making by revealing pathway activity in anatomically precise disease-affected regions.
Pipeline & Workflow Integration
Laser microdissection fits within the discovery continuum from hypothesis testing to lead identification, enabling spatially resolved molecular profiling that bridges imaging and omics.
- Discovery Biology: Supports hypothesis testing and pathway clarification by linking gene expression to structural landmarks in kidney tissue.
- Screening: Delivers assay readiness through high-success-rate dissection (>90%) and sufficient RNA yield for sequencing applications.
- Analytics: Provides quantitative dependent variable measurements (gene detection thresholds ≥10,000 genes) enabling cross-condition comparison.
- Translational Research: Advances preclinical continuity by preserving spatial context lost in dissociation-based methods.
- Enterprise Reuse: Functions as a reusable capability across tissue types via antibody marker substitution, supporting platform-wide adoption.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence by reducing mechanistic ambiguity in renal target validation.
- Operational Value: Enhances standardization and reproducibility through defined workflow parameters and QC checkpoints.
- Strategic Value: Improves go/no-go decisions by enabling risk-adjusted prioritization based on sub-segment-specific biology.
- Portfolio Impact: Supports capital efficiency through early de-risking of renal targets using spatially resolved evidence.
Implementation Considerations
- Requires expertise in immunofluorescence staining and morphological identification of renal sub-segments.
- Dependent on laser microdissection platforms and RNA isolation kits compatible with low-input samples.
- Necessitates cross-team standardization for staining protocols, dissection thresholds, and QC metrics.
- Adaptation across models requires validation of antibody markers and structural landmarks in target tissues.
- Practical limitations include user-dependent precision in region identification and RNA integrity maintenance during processing.
