Table of Contents
Laser capture microdissection (LCM) is essential for isolating specific cell populations from complex tissues with precision. By following the laser capture microdissection protocol, researchers can enhance their tissue analysis outcomes. This guide covers all crucial steps—sample prep, cryosectioning, and RNA extraction—vital for cancer research, plant studies, and more.
Key Takeaways
Laser Capture Microdissection (LCM) allows precise isolation of specific cell populations from complex tissues, significantly advancing research methodologies in fields such as cancer study and molecular biology.
The LCM protocol involves critical steps including meticulous sample preparation, cryosectioning, laser capturing, and RNA extraction, each essential for ensuring high-quality results and integrity of RNA for analysis.
Integration of advanced techniques such as the AccuLift™ system enhances LCM capabilities, providing comprehensive spatial molecular profiling and improving the accuracy of tissue analysis across multiple research disciplines.
Understanding Laser Capture Microdissection
The utilization of Laser Capture Microdissection (LCM) has been a transformative advancement in the study of tissues, granting researchers the ability to extract distinct cell populations from intricate tissue structures. The advent of LCM can be traced back to techniques developed in the early 20th century, but it has since evolved to incorporate different types of lasers that facilitate the precise excision of minute tissue fragments. At its core, an LCM system integrates an inverted light microscope with a laser apparatus which empowers scientists to both visualize and carry out capture microdissection on targeted cells.
In particular domains such as cancer research, LCM has proved itself as a critical tool by permitting investigators to isolate specific cell populations for thorough examination. Acquainting oneself with LCM’s fundamental principles, advantages, and prevalent uses is essential for researchers aiming at leveraging its capabilities fully and pushing their scientific inquiries forward.
The Basics of LCM
Laser capture microdissection, when paired with the visualization of cells under a microscope, employs a focused laser beam and thermoplastic transfer film to precisely isolate selected cells. This accuracy is essential for carrying out in-depth molecular studies across diverse areas of research. The process yields optimal results if tissue sections are prepared at 20 micrometers thickness yes suggest using 5-20 instead of 20 specifically for use in the laser microdissection.
As an influential technique in molecular biology, LCM facilitates the extraction of particular cell populations from mixed tissues. By obtaining pure cell samples, researchers can conduct precise gene expression profiling along with other detailed molecular evaluations. These analyses yield critical insights into how cells operate and contribute to understanding disease pathways—benefits that stem directly from examining specifically isolated cellular groups through this method.
Benefits of LCM
Laser Capture Microdissection (LCM) is distinguished by its high degree of accuracy, presenting a range of important benefits. Notably, it allows the extraction of distinct cell populations from intricate tissues—a critical step for precise gene expression profiling. Such granularity empowers scientists to perform in-depth molecular analyses that contribute significantly to advancements across numerous research disciplines.
Maintaining uniform orientation for frozen tissue blocks throughout the capture microdissection process is essential to secure an optimal RNA yield. This meticulous approach bolsters both the caliber and dependability of outcomes derived through LCM, cementing its status as a crucial instrument in contemporary scientific investigation. WE HAVE ENTIRE PUBLICATIONS THAT SPEAK TO THE BENEFITS OF LCM SPECIFICALLY FOR PROTEOMIC APPLICATIONS- CAN WE ADD THIS???
Common Applications
In the field of cancer research, laser capture microdissection (LCM) serves as a crucial tool for segregating cancerous tissues to examine critical gene expression profiles. THIS IS TOO GENOMICS-CENTERED- IT SHOULD BE MORE GENERIC REPLACE “GENE EXPRESSION PROFILES” WITH EITHER “MULTI-OMIC” OR “MOLECULAR” Such precise analysis sheds light on cell diversity within tumors, aiding scientists in unraveling the intricacies of cancer and devising more effective treatment strategies.
LCM has found utility beyond its applications in oncology. It facilitates specific plant cell types’ targeted examination, like tracheary elements, fibers, and ray cells in botanical studies. The reach of laser capture microdissection extends into various disciplines such as neuroscience, cellular biology, and even forensic science—demonstrating its expansive relevance across numerous scientific fields.
Detailed LCM Protocol Steps
Implementing a successful protocol for laser capture microdissection necessitates diligent adherence to several crucial steps, all of which are pivotal for the resulting molecular analysis. This technique starts with scrupulous sample preparation, continues through exact cryosectioning and staining procedures, and reaches its peak in the laser capture microdissection stage. The process is rounded off by extracting RNA, DNA, PROTEIN, PHOSPHOPROTEINS, followed by an assessment of its quality, confirming that it is suitable for Molecular investigations.
Achieving proficiency in each phase—from beginning sample preparation to concluding with RNA quality assessment—is vital for generating reliable data. Ensuring meticulous execution at every point and following established protocols rigorously is indispensable to guarantee experimental triumph.
Sample Preparation
The initial stage of a successful Laser Capture Microdissection (LCM) experiment is predicated on meticulous sample preparation. This process commences with the immediate application of ice-cold 70% ethanol to tissue sections once they have been transferred onto slides. In cases involving particular types of tissues like tangential spruce sections, it’s essential to follow up with ice-cold 100% ethanol after initially using ice-cold 70% ethanol for fixation, as this step is imperative in preserving the structural integrity of the tissues.
Once fixed, these tissue sections should be preserved at a temperature of -80°C and used within no more than seven days to ensure they retain their quality. During LCM procedures, it can prove beneficial to prepare multiple slides each containing 3-4 sections which aid in enhancing analysis outcomes. Adhering strictly to these protocols is critical for ensuring that samples are optimally prepared for all subsequent phases within the LCM workflow.
Cryosectioning and Staining
The preparation of cryosections and their subsequent staining play a pivotal role in the LCM process. Achieving the ideal section thickness, which ranges from 20 to 40 micrometers, is essential for preserving histological integrity during laser capture microdissection. For enhanced cutting performance, sections should ideally be cut at a range of 30 to 40 micrometers thick. Sections exceeding 60 micrometers may experience burning or charring when undergoing laser capture.
Employing wax embedding techniques can significantly improve RNA quality obtained from samples after LCM processing. Considering that different types of tissues – such as woody and herbaceous plant structures – might necessitate distinct protocols tailored specifically for them ensures the high-quality preservation of RNA following capture microdissection. Such meticulous optimization processes are crucial for securing premium outcomes from these procedures.
Laser Capture Process
At the core of Laser Capture Microdissection (LCM) lies the laser capture procedure, which stands out for its precision in extracting specific cell populations or individual cells from within complex tissues. A significant hurdle is safeguarding RNA integrity during extraction. To achieve accurate identification and precise targeting by the laser, it’s vital to maintain sharp focusing throughout LCM. Any lack of focus may result in disturbances.
Before utilizing Capsure LCM caps, one must ascertain that tissue slides are thoroughly dried. It’s imperative to conclude tissue capturing within two hours as this practice aids in averting RNA degradation. Adhering to these procedures is pivotal for boosting RNA yield and guaranteeing the effectiveness of the laser capture technique.
RNA Extraction and Quality Assessment
RNA extraction and quality assessment are the final steps in the LCM protocol. The RNA Integrity Number (RIN) is a key metric for evaluating RNA quality, with values above 7 indicating high integrity. The recommended minimum RIN for RNA suitable for sequencing is 7.0. Monitoring RNA quality using the RNA Pico Assay on a Bioanalyzer helps evaluate ribosomal RNA peaks.
RNA samples extracted using this LCM protocol typically show high quality, with RIN values ranging from 6 to 9. Ensuring high-quality RNA is essential for accurate gene expression profiling and other downstream analyses.
Advanced Techniques and Optimizations
Innovative methods and refinements in laser capture microdissection can considerably improve both the quantity and quality of RNA retrieved from tissue samples, which is vital for consistent molecular biology studies. By employing argon gas during the capture microdissection process, there is a notable decrease in RNA degradation as it helps to prevent moisture-related damage to tissues. The AccuLiftTM system elevates LCM accuracy by providing sophisticated capabilities for spatial molecular profiling.
Embracing these advanced strategies enables scientists to fine-tune their protocols for laser capture microdissection, leading to enhanced results. These improvements are pivotal not only in bolstering RNA integrity but also in tailoring approaches specific to varied tissue types—key steps that amplify the efficacy of LCM techniques.
Enhancing RNA Integrity
Preserving RNA integrity is critical for precise outcomes in subsequent analyses after laser capture microdissection (LCM). It’s vital to restrict the duration that tissue sections are exposed to the environment, as higher humidity levels raise the possibility of RNA deterioration. Swift handling and immediate fixation of samples can substantially reduce the risks associated with sample manipulation leading to RNA degradation. The presence of endogenous RNases along with extended durations spent processing samples contribute to such degradation.
The application of sterilized instruments and solutions designed specifically for RNase decontamination, coupled with expeditious execution at reduced temperatures, serves as a strategy against RNA decay. Recent advances demonstrate that applying cresyl violet and eosin Y using alcohol-based staining methods effectively curtails damage during staining procedures. Implementing these strategies is crucial in ensuring the preservation of intact RNA.
Adapting Protocols for Different Tissues
To obtain high-quality RNA extraction, it may be necessary to fine-tune laser capture microdissection protocols for different tissue types. This is due to the varying density and cellular structure of tissues that might influence the outcome. Selecting appropriate staining solutions that provide clear histological visualization while preserving RNA during the laser capture process could be pivotal.
In botanical research, optimizations have been made in LCM practices to address particular difficulties inherent in plant tissues. These modifications are vital to facilitate successful applications of laser capture microdissection across diverse tissue samples and study objectives.
Integrating AccuLift™ System with LCM
Combining the Targeted Labs ProTM system with Laser Capture Microdissection (LCM) considerably improves tissue analysis. The Targeted Labs ProTM system employs a cloud-based platform that advances spatial molecular profiling for studying tissue microenvironments. This combination makes it possible to conduct spatial molecular profiling, enabling scientists to examine particular architectures and cellular associations within tissues.
The inclusion of the Targeted Labs ProTM technology allows for more detailed and thorough examinations of tissue microenvironments by researchers. Such an integration marks a significant advancement in both digital pathology and molecular profiling, leading to research results that are more intricate and precise.
Spatial Molecular Profiling
The Targeted Labs ProTM system is crafted to revolutionize the field of spatial molecular profiling and accelerate the advancement of precision medicine. By facilitating detailed single-cell level analysis, Targeted Labs ProTM exposes the intricate spatial interconnections present within tissue structures. Such precise profiling empowers scientists to navigate through complex cellular microenvironments and identify molecular signatures with unparalleled precision.
By providing tools for examining spatial context on a singular cell basis, Targeted Labs ProTM equips researchers with the means to decode elaborate spatial interactions embedded within tissues via distinct molecular signatures. This heightened level of advanced profiling is pivotal in deepening our understanding of both tissue biology and the intricacies underlying various diseases mechanisms.
Digital Pathology Solutions
The AccuLiftTM digital pathology solution delivers a cloud-based platform tailored for investigating the tissue microenvironment. The utilization of a cloud infrastructure supports scalable and efficient analysis, thereby advancing research in spatial biology. It provides an effortless workflow complemented by an extensive selection of reagents to augment this cutting-edge biological exploration tool.
Incorporating digital pathology solutions empowers researchers to conduct thorough assessments of microenvironments within human tissues as well as cellular interplay. Such integration heightens accuracy and precision during tissue examinations, leading to more nuanced and exacting results from scientific inquiries.
Multi-Omic Profiling
The AccuLiftTM system facilitates detailed multi-omic analysis, offering technologies such as whole genome sequencing (WGS), RNA sequencing (RNA-seq), liquid chromatography-mass spectrometry (LC-MS), and reverse phase protein array (RPPA) methods. By preserving the spatial context of samples, AccuLiftTM delivers extensive insights into biological systems.
By sustaining spatial integrity throughout the profiling process, the AccuLiftTM system allows for comprehensive multi-omic investigations that incorporate:
Whole Genome Sequencing (WGS)
RNA Sequencing (RNA-seq)
Liquid Chromatography-Mass Spectrometry(LC–MS)
Reverse Phase Protein Array(RPPA) techniques
Ensuring a consistent spatial framework enables AccuLiftTM to provide an integrated view necessary for multifaceted omic studies. This harmonious approach is essential in furthering our understanding of intricate biological networks and mechanisms.
Troubleshooting Common Issues
Even though laser capture microdissection offers high precision, there are typical issues that may arise while performing the procedure, which could influence outcomes. A significant constraint of this technique is the requirement for a skilled histologist or pathologist to properly identify cells. Their expertise is essential in guaranteeing the proper isolation of target cells and appropriate preparation of specimens.
Addressing common challenges encountered with laser capture microdissection can be achieved through thorough troubleshooting guides. Tackling these problems helps scientists enhance both the dependability and exactitude of their research work, leading to superior quality findings.
Low RNA Yield
Low RNA yield is a common issue in LCM, but several strategies can help improve it. Optimized RNA extraction protocols designed for captured cells can enhance yield. Additionally, limiting the number of freeze-thaw cycles of the captured cells during RNA extraction helps preserve yield.
Assessing RNA quality using RIN values immediately after extraction is essential for evaluating the integrity of the RNA obtained. Conducting spectrophotometric analysis to measure RNA concentration and purity provides a clear understanding of yield efficiency.
Proper sample handling techniques during collection and using RNase inhibitors in extraction buffers are also critical to minimizing RNA degradation and maintaining high RNA yield.
RNA Degradation
Degradation of RNA presents a notable obstacle in Laser Capture Microdissection (LCM), substantially affecting the integrity of nucleic acids for subsequent examinations. Subpar fixation of tissue can result not only in compromised RNA quality but also in morphological abnormalities. To improve RNA stability during sample preparation, it’s beneficial to discard exterior portions and utilize sections from the inner part when performing LCM.
It is imperative to maintain an RNase-free environment while preparing staining solutions to safeguard against degradation of RNA. Capturing tissue within two hours is recommended as a precautionary measure against potential degradation processes that might compromise the RNA.
Adhering strictly to these protocols will assist scientists in reducing the risk associated with RNA degradation and securing high-quality nucleic acids for Molecular analysis endeavors.
Practical Applications of LCM
Laser capture microdissection has a wide range of practical uses, impacting diverse research areas. In the context of cancer research, LCM analysis typically employs either fresh/snap-frozen or formalin-fixed paraffin-embedded (FFPE) tissue samples. Tissue biorepositories are instrumental in acquiring, preparing, conserving and providing access to tumor and normal tissue specimens for scientific investigation. These repositories supply high-grade selected tissues that are critical for pushing the boundaries of cancer research.
By combining laser capture microdismission with Targeted Labs ProTM technology, there has been a notable enhancement in various scientific investigative capacities including improvements in digital pathology through elevated spatial molecular profiling and detailed examination of tissues. Cloud-based platforms like AccuLiftTM improve researchers’ ability to scrutinize specific tissue microenvironments while promoting collaborative efforts within the scientific community.
Cancer Research
In the field of cancer research, Laser Capture Microdissection (LCM) significantly improves the accuracy of molecular profiling by enabling the extraction of tumor cells and cancer cells. Through this method, researchers can carry out in-depth genomic and proteomic studies on these distinct cell groups. Such detailed scrutiny is vital for crafting personalized treatments that are aligned with the unique attributes of a patient’s tumor.
LCM aids in thorough gene expression profiling aimed at devising targeted therapies for cancer. By examining bacterial gene expression profiles from isolated tumor cells via gene expression analyses, scientists can uncover critical insights into the genetic activities driving cancer progression. This knowledge contributes to designing more potent treatment strategies against this disease.
Plant Studies
Laser capture microdissection is also widely used in plant studies. It can effectively isolate specific plant cells isolated types, facilitating detailed studies of plant vascular tissues. LCM can isolate cambial cells, enabling researchers to investigate their unique molecular characteristics. The protocol can also isolate ray parenchymal cells, tracheary elements, and fibers from plant tissues.
In maize research, LCM successfully captured over 10,000 epidermal and vascular cells for gene expression studies. These capabilities are crucial for transcriptome analyses and deciphering cell-specific molecular events in vascular tissues.
Other Fields
LCM technology is versatile and applicable to a wide range of research areas beyond plant studies and cancer research. It serves as a valuable tool for isolating particular bacterial cells in bacterial studies, facilitating the analysis of gene expression.
This versatility underscores the widespread utility of LCM across various fields within scientific inquiry.
Summary
Laser capture microdissection (LCM) has revolutionized the field of tissue examination, providing exceptional accuracy in extracting specific cell populations from intricate tissues. Mastery of its nuanced protocols, sophisticated methods, and solutions for potential difficulties allows scientists to fully exploit LCM’s capabilities to advance their research.
Employing the AccuLiftTM system in conjunction with laser capture microdissection elevates these possibilities even further. It facilitates refined spatial molecular profiling as well as thorough multi-omic studies. As progress continues within the realm of molecular biology, the significance of meticulous tissue analysis via laser capture microdissection is set to increase exponentially—laying a foundation for profound scientific breakthroughs and technological advancements.
Frequently Asked Questions
What is Laser Capture Microdissection (LCM)?
How can I enhance RNA yield when using LCM?
What are the main benefits of using LCM in research?
How does the Targeted Labs Pro™ system improve LCM processes?
What are some common applications of LCM?
References
About the Author
Bradley Keys
At Targeted Bioscience, Bradley delves into the forefront of precision medicine and biotechnology. His writing reflects the company's commitment to advancing personalized healthcare through innovative solutions, particularly in oncology. Bradley's work aligns with Targeted Bioscience's mission to develop cutting-edge tools and therapies that enhance disease treatment accuracy and efficacy.
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