Laser Capture Microdissection: Understanding the Techniques and Implications for Molecular Biology in Nursing Research Through Analysis of Breast Cancer Tumor Samples

2011 ◽  
Vol 13 (3) ◽  
pp. 297-305 ◽  
Author(s):  
Karen L. Zanni ◽  
Garrett K. Chan
Keyword(s):  
Laser Capture Microdissection ◽  
Breast Biopsy ◽  
Ductal Carcinoma ◽  
Pcr Amplification ◽  
Cell Types ◽  
Cell Populations ◽  
Specific Cell ◽  
Pure Cell ◽  
Heterogeneous Tissue ◽  
Laser Capture

Aim: The purpose of this paper is to review the techniques and implications of laser capture microdissection (LCM) to isolate tissue and DNA of interest using breast biopsy tissue as an example. Background: Tissues are a heterogeneous mix of different cell types, and molecular alterations are often specific to a single cell type. An accurate correlation of molecular and morphologic pathologies requires the ability to procure pure populations of morphologically similar cells for molecular analysis. LCM is a technique for isolating highly pure cell populations of morphologically similar cells from a heterogeneous tissue section. Method: Nine invasive, paraffin-embedded breast biopsy specimens were obtained and analyzed. Depending on the size of the lesion, 500—1,000 shots using the 7.5- or 15-µm infrared laser beam were utilized to obtain an average of 2,000 cells. DNA was isolated from normal tissue and carcinomas and polymerase chain reaction (PCR) amplification was examined by agarose gel electrophoresis. The HER2/neu gene was amplified by standard PCR. A second round of PCR using nested primers to re-amplify the HER2/neu fragment was performed. Results: Amplification of the HER2/neu gene with DNA isolated from pure cell populations by LCM was performed. The results indicated that 22% of the cases studied were positive for HER2/neu amplifications, which corresponds to the literature regarding HER2/neu amplification/overexpression. HER2/neu amplification could be detected as early as the ductal carcinoma in situ (DCIS) stage. Conclusion: LCM is an accurate and reliable method to acquire nucleic acid and protein profiles from a specific cell population in heterogeneous tissue.

scholarly journals Laser Capture Microdissection in Dentistry

2010 ◽  
Vol 2010 ◽  
pp. 1-8 ◽  
Author(s):  
Uraiwan Chokechanachaisakul ◽  
Tomoatsu Kaneko ◽  
Takashi Okiji ◽  
Reika Kaneko ◽  
Hideaki Suda ◽  
...  
Keyword(s):  
Laser Capture Microdissection ◽  
Gene Expression Analysis ◽  
Cell Types ◽  
Specific Cell ◽  
Tissue Sections ◽  
Rna Analysis ◽  
Molecular Biological Analysis ◽  
Polymerase Chain ◽  
Tissue Specimens ◽  
Laser Capture

Laser capture microdissection (LCM) allows for the microscopic procurement of specific cell types from tissue sections that can then be used for gene expression analysis. According to the recent development of the LCM technologies and methodologies, the LCM has been used in various kinds of tissue specimens in dental research. For example, the real-time polymerase-chain reaction (PCR) can be performed from the formaldehyde-fixed, paraffin-embedded, and immunostained sections. Thus, the advance of immuno-LCM method allows us to improve the validity of molecular biological analysis and to get more accurate diagnosis in pathological field in contrast to conventional LCM. This paper is focused on the presentation and discussion of the existing literature that covers the fields of RNA analysis following LCM in dentistry.

scholarly journals Probe-Seq enables transcriptional profiling of specific cell types from heterogeneous tissue by RNA-based isolation

2019 ◽  
Author(s):  
Ryoji Amamoto ◽  
Mauricio D. Garcia ◽  
Emma R. West ◽  
Jiho Choi ◽  
Sylvain W. Lapan ◽  
...  
Keyword(s):  
Transcriptional Profiling ◽  
Cell Types ◽  
Cell Populations ◽  
Specific Cell ◽  
Rna Profiling ◽  
Complementary Method ◽  
Dissociated Cells ◽  
Multiple Cell ◽  
Heterogeneous Tissue

ABSTRACTRecent transcriptional profiling technologies are uncovering previously-undefined cell populations and molecular markers at an unprecedented pace. While single cell RNA (scRNA) sequencing is an attractive approach for unbiased transcriptional profiling of all cell types, a complementary method to isolate and sequence specific cell populations from heterogeneous tissue remains challenging. Here, we developed Probe-Seq, which allows deep transcriptional profiling of specific cell types isolated using RNA as the defining feature. Dissociated cells are labelled using fluorescent in situ hybridization (FISH) for RNA, and then isolated by fluorescent activated cell sorting (FACS). We used Probe-Seq to purify and profile specific cell types from mouse, human, and chick retinas, as well as the Drosophila midgut. Probe-Seq is compatible with frozen nuclei, making cell types within archival tissue immediately accessible. As it can be multiplexed, combinations of markers can be used to create specificity. Multiplexing also allows for the isolation of multiple cell types from one cell preparation. Probe-Seq should enable RNA profiling of specific cell types from any organism.

scholarly journals Optimizing Laser Capture Microdissection Protocol for Isolating Zone-Specific Cell Populations from Mandibular Condylar Cartilage

2019 ◽  
Vol 2019 ◽  
pp. 1-13
Author(s):  
Aisha M. Basudan ◽  
Yanqi Yang
Keyword(s):  
Laser Capture Microdissection ◽  
Genetic Data ◽  
Male Rats ◽  
Cell Populations ◽  
Specific Cell ◽  
Condylar Cartilage ◽  
Rna Integrity ◽  
Tissue Sections ◽  
Mandibular Condylar Cartilage ◽  
Laser Capture

Mandibular condylar cartilage (MCC) is a multizonal heterogeneous fibrocartilage consisting of fibrous (FZ), proliferative (PZ), mature (MZ), and hypertrophic (HZ) zones. Gross sampling of the whole tissue may conceal some important information and compromise the validity of the molecular analysis. Laser capture microdissection (LCM) technology allows isolating zonal (homogenous) cell populations and consequently generating more accurate molecular and genetic data, but the challenges during tissue preparation and microdissection procedures are to obtain acceptable tissue section morphology that allows histological identification of the desirable cell type and to minimize RNA degradation. Therefore, our aim is to optimize an LCM protocol for isolating four homogenous zone-specific cell populations from their respective MCC zones while preserving the quality of RNA recovered. MCC and FCC (femoral condylar cartilage) specimens were harvested from 5-week-old Sprague–Dawley male rats. Formalin-fixed and frozen unfixed tissue sections were prepared and compared histologically. Additional specimens were microdissected to prepare LCM samples from FCC and each MCC zone individually. Then, to evaluate LCM-RNA integrity, 3′/m ratios of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin (β-Actin) using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were calculated. Both fixed and unfixed tissue sections allowed reliable identification of MCC zones. The improved morphology of the frozen sections of our protocol has extended the range of cell types to be isolated. Under the empirically set LCM parameters, four homogeneous cell populations were efficiently isolated from their respective zones. The 3′/m ratio means of GAPDH and β-Actin ranged between 1.11–1.56 and 1.41–2.12, respectively. These values are in line with the reported quality control requirements. The present study shows that the optimized LCM protocol could allow isolation of four homogenous zone-specific cell populations from MCC, meanwhile preserving RNA integrity to meet the high quality requirements for subsequent molecular analyses. Thereby, accurate molecular and genetic data could be generated.

scholarly journals Probe-Seq enables transcriptional profiling of specific cell types from heterogeneous tissue by RNA-based isolation

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Ryoji Amamoto ◽  
Mauricio D Garcia ◽  
Emma R West ◽  
Jiho Choi ◽  
Sylvain W Lapan ◽  
...  
Keyword(s):  
Transcriptional Profiling ◽  
Cell Types ◽  
Cell Populations ◽  
Specific Cell ◽  
Rna Profiling ◽  
Complementary Method ◽  
Dissociated Cells ◽  
Multiple Cell ◽  
Heterogeneous Tissue

Recent transcriptional profiling technologies are uncovering previously-undefined cell populations and molecular markers at an unprecedented pace. While single cell RNA (scRNA) sequencing is an attractive approach for unbiased transcriptional profiling of all cell types, a complementary method to isolate and sequence specific cell populations from heterogeneous tissue remains challenging. Here, we developed Probe-Seq, which allows deep transcriptional profiling of specific cell types isolated using RNA as the defining feature. Dissociated cells are labeled using fluorescent in situ hybridization (FISH) for RNA, and then isolated by fluorescent activated cell sorting (FACS). We used Probe-Seq to purify and profile specific cell types from mouse, human, and chick retinas, as well as from Drosophila midguts. Probe-Seq is compatible with frozen nuclei, making cell types within archival tissue immediately accessible. As it can be multiplexed, combinations of markers can be used to create specificity. Multiplexing also allows for the isolation of multiple cell types from one cell preparation. Probe-Seq should enable RNA profiling of specific cell types from any organism.

scholarly journals Chemogenetic Tools for Causal Cellular and Neuronal Biology

2018 ◽  
Vol 98 (1) ◽  
pp. 391-418 ◽  
Author(s):  
Deniz Atasoy ◽  
Scott M. Sternson
Keyword(s):  
G Protein ◽  
Ex Vivo ◽  
Cell Types ◽  
Cell Populations ◽  
Specific Cell ◽  
Cellular Processes ◽  
Distinct Cell ◽  
G Protein Coupled ◽  
Pharmacological Control

Chemogenetic technologies enable selective pharmacological control of specific cell populations. An increasing number of approaches have been developed that modulate different signaling pathways. Selective pharmacological control over G protein-coupled receptor signaling, ion channel conductances, protein association, protein stability, and small molecule targeting allows modulation of cellular processes in distinct cell types. Here, we review these chemogenetic technologies and instances of their applications in complex tissues in vivo and ex vivo.

scholarly journals Localization of ACE2 in the renal vasculature: amplification by angiotensin II type 1 receptor blockade using telmisartan

2009 ◽  
Vol 296 (2) ◽  
pp. F398-F405 ◽  
Author(s):  
María José Soler ◽  
Minghao Ye ◽  
Jan Wysocki ◽  
Josette William ◽  
Josep Lloveras ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Laser Capture Microdissection ◽  
Receptor Blocker ◽  
Specific Cell ◽  
Renal Vasculature ◽  
Tunica Media ◽  
Quantitative Measurements ◽  
Mrna Ratio ◽  
Laser Capture

Angiotensin-converting enzyme (ACE)2 is a carboxypeptidase that degrades angiotensin II and other peptides. In the kidney, ACE2 localization within the glomerulus and tubules is cell specific. This study was aimed to investigate the localization of ACE2 within the renal vasculature. We also studied the effect of the administration of a specific angiotensin II type 1 receptor blocker, telmisartan, on ACE2 expression in the renal vasculature. ACE2 and ACE were localized in renal arterioles using confocal microscopy and specific cell markers. Quantitative measurements of ACE2 and ACE mRNA were estimated in kidney arterioles isolated by laser capture microdissection using real-time PCR. In kidney arterioles, ACE was localized in the endothelial layer, whereas ACE2 was localized in the tunica media. In mice treated with telmisartan (2 mg·kg−1·day−1) for 2 wk, ACE2 expression was increased by immunostaining, whereas ACE expression was decreased. This was reflected in a decrease in the ACE/ACE2 ratio compared with vehicle-treated controls (0.53 ± 0.14 vs. 7.59 ± 2.72, P = 0.027, respectively). In kidney arterioles isolated by laser capture microdissection, the ACE/ACE2 mRNA ratio was also decreased compared with control mice (1.21 ± 0.31 vs. 4.63 ± 0.86, P = 0.044, respectively). In conclusion, in kidney arterioles ACE2 is preferentially localized in the tunica media, and its expression is increased after administration of the angiotensin II type 1 receptor blocker, telmisartan. Amplification of ACE2 in the renal vasculature may contribute to the therapeutic action of telmisartan by increasing angiotensin II degradation.

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