Detection of MicroRNA Expression in the Kidneys of Immunoglobulin A Nephropathic Mice
July 8th, 2020
microRNAs are involved in the pathogenesis of IgA nephropathy. We have developed a reliable method for detecting microRNA expression levels in the kidneys of an IgA nephropathy mouse model (HIGA mice). This new method will facilitate to check for miRNAs involvement in IgA nephropathy.
This method can be used to easily verify the change of microRNAs in IGA nephropathy using a small animal model, making it possible to study IGA nephropathy without a human kidney biopsy. The protocol can provide new insights into the genetics of IGA nephropathy, which can potentially aid in discovery of new diagnostic and new therapeutic methods for the kidney disease. After ensuring that the mouse is properly anesthetized, use surgical scissors and forceps to make a three-to four-centimeter midline incision in the abdominal wall of the mouse and identify both sides of the kidney.
Incise the ribs and diaphragm to expose the heart. After incising the right atrium, inject PBS into the left ventricle until the kidney color changes to pale yellow, indicating that the whole body of the mouse is perfused with PBS. Cut the renal artery, renal vein, and ureter, then remove the kidney.
Homogenize the 30-milligram kidney samples using a silicon homogenizer and 700 microliters of phenol guanidine-based lysis reagent at room temperature. Transfer the lysate to the biopolymer shredding spin column and centrifuge it at 15, 000 G for two minutes at room temperature. Continue with RBNA purification following manuscript directions.
After the final wash, add 30 microliters of nuclease-free water to the silica membrane-based spin column and centrifuge it at 8, 000 G for one minute to elute the RNA. For cDNA synthesis, prepare a master mix with two microliters of nucleic acid mix, two microliters of reverse transcriptase mix, and four microliters of buffer in a total of eight microliters per sample. Add eight microliters of the master mix to each well of an eight-well tube strip.
Dilute total RNA in nuclease-free water. Then, add 12 microliters of the total RNA solution to each well of the strip. Place the samples in the thermocycler and incubate them at 37 degrees Celsius for 60 minutes, then at 95 degrees Celsius for five minutes.
To perform quantitative real-time PCR, prepare the master mix according to manuscript directions, then add 22.5 microliters of the master mix to each well of a 96-well reaction plate. Add 2.5 microliters of the prepared cDNA to each well. Place the reaction plate in the real-time PCR instrument.
Then, program it for an initial activation at 95 degrees Celsius for 15 minutes, followed by 40 cycles of denaturation at 94 degrees Celsius for 15 seconds, annealing at 55 degrees Celsius for 30 seconds, and extension at 70 degrees Celsius for 30 seconds. Quantify gene expression using the delta-delta CT method. This protocol was used to investigate the expression levels of microRNAs in the kidneys of HIGA mice using the kidneys of Balb/c mice as control.
The expression levels of three IGA nephropathy-associated microRNAs were measured. Relative microRNA expression levels of the two groups were compared using a T-test with a P of less than 0.05 being statistically significant. miR-155-5p was expressed at 3.3-fold higher levels in the HIGA group compared with the control group, and miR-21-5p was expressed at 1.58-fold higher levels.
miR-1461-5p expression did not differ significantly between the two groups. When attempting this protocol, it is important to wear a mask, gloves, and cap and to keep the laboratory table and tools clean in order to prevent nuclease in the environment from affecting the experiment. This experimental method is currently in practical use in our laboratory.
It's goal, to establish new diagnostic and therapeutic methods for IGA nephropathy.
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Overview
This study presents a reliable method for detecting microRNA expression levels in the kidneys of an IgA nephropathy mouse model. The method allows for the investigation of microRNAs' involvement in IgA nephropathy without the need for human kidney biopsies.
Key Study Components
Area of Science
- Nephrology
- MicroRNA research
- Animal models
Background
- IgA nephropathy is a kidney disease characterized by the deposition of IgA in the glomeruli.
- MicroRNAs play a significant role in the pathogenesis of various diseases, including kidney disorders.
- Understanding microRNA expression can provide insights into disease mechanisms.
- Current methods for studying microRNAs often require invasive procedures.
Purpose of Study
- To develop a method for assessing microRNA levels in a mouse model of IgA nephropathy.
- To facilitate research on the genetic aspects of IgA nephropathy.
- To explore potential diagnostic and therapeutic approaches for kidney disease.
Methods Used
- Use of HIGA mice as a model for IgA nephropathy.
- Administration of anesthesia to the mouse prior to surgery.
- Surgical techniques to access the kidneys, including midline incision and organ removal.
- Perfusion of the mouse with PBS to prepare for kidney extraction.
Main Results
- The developed method allows for easy verification of microRNA changes in IgA nephropathy.
- Insights gained from this method can lead to new diagnostic tools.
- Potential for discovering new therapeutic strategies for treating IgA nephropathy.
- Elimination of the need for human kidney biopsies in preliminary studies.
Conclusions
- This method enhances the understanding of microRNA involvement in IgA nephropathy.
- It opens new avenues for research into kidney disease genetics.
- The approach may significantly impact future diagnostic and therapeutic developments.
Frequently Asked Questions
Executive Industry Relevance
This protocol enables biopharma R&D teams to profile microRNA expression in a validated IgA nephropathy mouse model, supporting target validation and mechanistic de-risking in kidney disease programs. By providing a reproducible method to detect altered miRNA levels without human biopsies, it facilitates early discovery of biomarkers and therapeutic targets with improved predictive confidence. The approach aligns with discovery-stage efforts to interrogate disease mechanisms and prioritize candidates for preclinical development.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses by quantifying disease-associated miRNA expression changes in HIGA mice versus controls.
- Operational Value: Provides a reliable workflow for functional target validation using qRT-PCR to measure miR-155-5p, miR-21-5p, and miR-146a-5p levels.
- Predictive Value: Supports portfolio triage by identifying miRNAs with statistically significant fold-changes (e.g., 3.3-fold for miR-155-5p) as potential biomarkers or targets.
Screening & Assay Development
- Scientific Value: Delivers quantitative, reproducible miRNA expression data suitable for assay standardization in discovery screening campaigns.
- Operational Value: Establishes a standardized RNA purification and cDNA synthesis protocol compatible with high-throughput qRT-PCR platforms.
- Assay Readiness: Generates normalized CT values using the delta-delta CT method, enabling cross-sample comparison and hit confirmation.
Translational & Preclinical Research
- Translational Value: Uses a disease-relevant HIGA mouse model to bridge discovery findings with preclinical validation of miRNA-mediated pathways.
- Mechanistic De-risking: Allows assessment of miRNA expression changes linked to IgA deposition, reducing ambiguity in target mechanism prior to lead identification.
- Preclinical Continuity: Supports risk-adjusted advancement decisions by providing consistent, quantifiable readouts from kidney tissue across study groups.
Pipeline & Workflow Integration
The method fits within the early discovery continuum, supporting hypothesis testing in target validation and generating quantitative outputs for screening and analytics workflows that inform lead identification decisions.
- Discovery Biology: Enables pathway clarification by measuring miRNA expression shifts associated with IgA nephropathy pathogenesis in a genetic mouse model.
- Screening: Produces standardized, quantitative miRNA data suitable for evaluating compound effects on target pathways in downstream assays.
- Analytics: Generates delta-delta CT values and fold-change metrics that allow teams to compare conditions and assess statistical significance using T-tests.
- Translational Research: Connects discovery-phase miRNA findings to preclinical continuity through use of a validated IgA nephropathy model with phenotypic relevance.
- Enterprise Reuse: Establishes a reusable RNA extraction and qRT-PCR protocol applicable to multiple miRNA targets across kidney disease projects.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in target validation by reducing mechanistic ambiguity through direct measurement of miRNA dysregulation in a disease model.
- Operational Value: Ensures reproducibility and standardization via defined RNA purification, cDNA synthesis, and qRT-PCR parameters (e.g., 8,000 G centrifugation, 37°C incubation).
- Strategic Value: Improves go/no-go decisions by providing statistically significant expression data (p<0.05) to prioritize targets with strong disease association.
- Portfolio Impact: Enables risk-adjusted prioritization of miRNA-based biomarkers or therapeutic targets based on fold-change magnitude and reproducibility.
Implementation Considerations
- Requires expertise in rodent surgery, RNA handling, and qRT-PCR to prevent nuclease degradation and ensure sample integrity.
- Depends on access to homogenization equipment, centrifuge, thermocycler, and real-time PCR instrument with fluorescence detection.
- Necessitates standardization across teams for RNA input (30 mg kidney), reagent volumes (700 μL lysis reagent), and cycling conditions (95°C activation, 40 cycles).
- Requires adaptation of primer sets for specific miRNA targets while maintaining consistent annealing (55°C) and extension (70°C) temperatures.
- Limited to endpoint RNA analysis; does not provide spatial or single-cell resolution within kidney tissue without additional methodologies.
