Laser Capture Microdissection: Beyond Functional Genomics to Proteomics

doi:10.1054/modi.2000.19808a

Molecular Diagnosis

Volume 5, Issue 4, December 2000, Pages 301-307

https://doi.org/10.1054/modi.2000.19808a Get rights and content

Proteomics will drive biology and medicine beyond genomics, and can have a profound impact on molecular diagnostics. The posttranslational modifications of cellular proteins that govern physiology and become deranged in disease cannot be accurately portrayed by gene expression alone. Consequently, new technology is being developed to discover, and quantitatively monitor, proteomic changes that are associated with disease etiology and progression. In the past, proteomic technologies were restricted to tumor cell lines or homogenized bulk tissue specimens. This source material may not accurately reflect molecular events taking place in the specific cells of the tissue itself. This article describes a completely new class of proteomic-based approaches aimed at the identification and investigation of protein markers in the actual histologically defined cell populations that are immersed in heterogeneous diseased tissue. It is envisioned that these investigations will eventually lead to novel diagnostic, prognostic, or therapeutic markers that can be applied to monitor therapeutic toxicity or efficacy.

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Cited by (93)

Role of proteomics in surgical oncology
2023, Proteomics: A Promising Approach for Cancer Research
The molecular sciences currently place more emphasis on proteomics since it provides crucial information on protein identification, expression levels, and modification. For instance, cancer proteomics has aided in the discovery of crucial information in mechanistic studies on the growth and metastasis of tumors, leading to the creation of clinically valuable biomarkers and therapeutic targets. Researchers from all over the world can access a variety of cancer proteome datasets. Proteomics and other omics studies should be combined since they provide a plethora of knowledge on biological pathways and target stimulators. Surgical research includes studies that link genomic findings with a patient’s clinical outcome, clinically based pathophysiological research, studies of the therapeutic response, etc. This book chapter discusses proteomic technologies and gives the most recent review of studies that have looked at cancer biomarkers using these technologies. Even if the current results are positive, additional reliable research in multicenter networks is required to help translate omics from the lab to the clinic. In the modern era, proteomics plays a crucial part in surgical oncology; thus, it is important to be aware of the cancer detection and numerous updates about the cell. For this reason, approaches that can be patient-friendly are needed to support surgical oncology. The impact of this strategy on the patients’ health was enormous. We have discussed the widely used proteome technologies in this book chapter in order to better understand the pathophysiology of various cancers and conduct surgical proteomic research. It may ultimately lead to fewer fatalities and the advancement of molecular diagnostics, which can be used to detect cancer early.
A simple, cost-effective and flexible method for processing of snap-frozen tissue to prepare large amounts of intact RNA using laser microdissection
2012, Biochimie
Citation Excerpt :
The lack of RNA integrity in such studies has been criticized at best as unreliable and at worst as misleading [3–5]. The use of laser microdissection (LMD) [6] has transformed the acquisition of homogeneous cell populations for basic science [7–10], forensic science [1,2,11] and molecular medicine [7,12,13]. However, the quality and quantity of RNA recovered from samples obtained using this technique is quite low.
Understanding the molecular basis of disease requires gene expression profiling of normal and pathological tissue. Although the advent of laser microdissection (LMD) has greatly facilitated the procurement of specific cell populations, often only small amounts of low quality RNA is recovered. This precludes the use of global approaches of gene expression profiling which require sizable amounts of high quality RNA. Here we report a method for processing of snap-frozen tissue to prepare large amounts of intact RNA using LMD.
Portions of small intestine from piglets (n = 6) were snap-frozen in Optimum Cutting Temperature compound (experimental) and in RNAlater (control). A randomly selected sample was laser microdissected using the developed protocol in multiple sessions totalling 4 h each day on four consecutive days. RNAs were extracted from these samples and its control and their quality (RIN) determined. RINs of the experimental samples were independent of time (p = 0.12) and day (p = 0.56) of the microdissection thereby suggesting that their RNA quality remained unaltered. These samples exhibited high quality (RIN ≥ 8) with good recovery (81.2%) and excellent yield (1539 ng/1.2 × 10⁷ μm²). Their overall RIN, 8.029 ± 0.116, was not significantly different from 8.2 (p = 0.123), the value obtained from the control, non-laser microdissected, sample. This indicated that the RNA quality from the laser microdissected and non-microdissected samples was comparable.
The method allowed LMD for up to 4 h each day for a total of four days. The microdissected samples can be pooled thereby increasing amount of RNA at least by ten-fold. The procedure did not require any expensive limited-shelf life RNase inhibitors, RNA protectors, staining kits or toxic chemicals. Furthermore, it was flexible and enabled the processing without affecting routine laboratory workflow.
The method developed was simple, inexpensive and provided substantial amounts of high quality RNA suitable for gene expression profiling and other cellular and molecular analyses for biology and molecular medicine.
Nutriproteomics: A promising tool to link diet and diseases in nutritional research
2012, Biochimica et Biophysica Acta - Proteins and Proteomics
Citation Excerpt :
Tissue culture samples treated with different compounds can be analyzed for their proteomes by the above described means. However, an analysis with variations in the biological sample such as different combinations of cell types can involve an initial separation of the different cells using cell specific surface markers and immunoaffinity techniques or laser driven microdissection approaches [26,139]. Apart from the analytical complexity, there are other limitations such as cost, resolution, reproducibility and throughput [5].
Nutriproteomics is a nascent research arena, exploiting the dynamics of proteomic tools to characterize molecular and cellular changes in protein expression and function on a global level as well as judging the interaction of proteins with food nutrients. As nutrients are present in complex mixtures, the bioavailability and functions of each nutrient can be influenced by the presence of other nutrients/compounds and interactions. The first half of this review focuses on the techniques used as nutriproteomic tools for identification, quantification, characterization and analyses of proteins including, two-dimensional polyacrylamide electrophoresis, chromatography, mass spectrometry, microarray and other emerging technologies involving visual proteomics. The second half narrates the potential of nutriproteomics in medical and nutritional research for revolutionizing biomarker and drug development, nutraceutical discovery, biological process modeling, preclinical nutrition linking diet and diseases and structuring ways to a personalized nutrition. Though several challenges such as protein dynamics, analytical complexity, cost and resolution still exist, the scope of applying proteomics to nutrition is rapidly expanding and promising as more holistic strategies are emerging.
Protein analysis through Western blot of cells excised individually from human brain and muscle tissue
2012, Analytical Biochemistry
Comparing protein levels from single cells in tissue has not been achieved through Western blot. Laser capture microdissection allows for the ability to excise single cells from sectioned tissue and compile an aggregate of cells in lysis buffer. In this study we analyzed proteins from cells excised individually from brain and muscle tissue through Western blot. After we excised individual neurons from the substantia nigra of the brain, the accumulated surface area of the individual cells was 120,000, 24,000, 360,000, 480,000, 600,000 μm². We used an optimized Western blot protocol to probe for tyrosine hydroxylase in this cell pool. We also took 360,000 μm² of astrocytes (1700 cells) and analyzed the specificity of the method. In muscle we were able to analyze the proteins of the five complexes of the electron transport chain through Western blot from 200 human cells. With this method, we demonstrate the ability to compare cell-specific protein levels in the brain and muscle and describe for the first time how to visualize proteins through Western blot from cells captured individually.
Prostate-specific antigen mRNA and protein levels in laser microdissected cells of human prostate measured by real-time reverse transcriptase-quantitative polymerase chain reaction and immuno-quantitative polymerase chain reaction
2008, Human Pathology
Citation Excerpt :
An additional advantage is that LAM allows molecular analysis of cell populations in their native tissue environment and may be potentially applicable to biopsies obtained in preoperative diagnostic procedures. This new technique can thus overcome the problem of cellular heterogeneity that is a significant barrier to the molecular analysis of normal versus pathological tissues [10]. So far, few studies have reported on the protein content of microdissected tissue samples [11], mainly because of the limited sensitivity of protein immunoassays.
Laser-assisted microdissection has mainly been used in cancer studies to excise pure cell populations from heterogeneous tissues. Cancer and normal cells selected by laser-assisted microdissection have frequently been used for mRNA expression studies usually by reverse transcriptase–quantitative polymerase chain reaction (qPCR). Recently, real time immuno-qPCR was developed as a new tool for highly sensitive measurements of proteins. Using reverse transcriptase–qPCR and immuno-qPCR, we measured the amounts of prostate-specific antigen mRNA and its corresponding protein in homogeneous and comparable cell populations, collected from normal and cancer prostates by laser-assisted microdissection. With these techniques, prostate-specific antigen mRNA and protein were quantified over a wide range of concentrations with a sensitivity sufficient to analyze single prostate cells (LNCaP). We did not find significant differences in prostate-specific antigen protein and mRNA between normal and cancer cells. The expression of prostate-specific antigen protein and mRNA was highly correlated in both normal and pathological cells. In microdissected peritubular stromal areas of prostate cancers, the concentration of prostate-specific antigen protein was about 100 times higher than in normal prostate, indicating an increased transit of secreted prostate-specific antigen. In the same samples, prostate-specific antigen mRNA was not detectable. Our data demonstrate, for the first time, the feasibility of simultaneous application of reverse transcriptase–qPCR and immuno-qPCR in studies of homogeneous cell populations, collected by laser-assisted microdissection. The approach is expected to become a very powerful tool for expression studies in human cancers at both mRNA and protein levels.
Proteomics: Methodologies and Applications in Oncology
2008, Seminars in Radiation Oncology
Citation Excerpt :
However, because tumors are characterized by both tumor cells and a range of host-derived cells, careful consideration must be made with respect to the starting material for comparison-based proteomic profiling. One approach is to use laser-capture microdissection to identify the areas within the tumor of interest and then to isolate the proteins specifically from this area before proteomic analysis with MS. This approach was first used to compare early- and late-stage disease to identify proteins associated with tumor progression using the SELDI machine.43 Although very few cells are required (as few as 25 will create a suitable MS peptide spectra), this is a rather tedious procedure and not easily amenable to high-throughput approaches.
Few technological developments have created as much excitement and skepticism as proteomics over their potential to change clinical diagnostic and prognostic procedures. Proteomics concerns itself with the characterization and function of all cellular proteins, the ultimate determinants of cellular function. As such, it represents the end result of all mechanisms of gene regulation and thus offers tremendous potential for characterizing biology. In much the same way as what has occurred with the genome, the scientific community is coming to grips with the fact that the proteome, although enormously complex, is finite. It is conceivable that we will learn the identity of all possible proteins, including all posttranslational modifications. The rate of protein discovery continues to accelerate in large part because of improvements in mass spectrometry–based technologies coupled with improved genomic databases and bioinformatic tools. In addition, there is reason to believe that proteomics is on the verge of moving from a methodology that requires repeated proteome “discovery” to one that can more systematically profile proteomes. This review discusses current proteomic-based technologies and the efforts of scientists to move them into the clinic for use in patients treated with radiotherapy and other modalities.

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