Using Lyve1-Cre+ Mice to Visualize Lymphatics for Laser Capture Microdissection

Background: Lymphedema is characterized by limb swelling secondary to lymphatic dysfunction. Lymphedema most frequently develops following breast cancer treatment due to iatrogenic damage of the lymphatics from surgery and radiation. Lymphedema affects 20-40% of breast cancer survivors. There is no cure for this disease. For determination of successful delivery of gene-based therapies, target cells are often isolated and analyzed via real-time PCR. One method to isolate a region of cells within a tissue section is laser capture microdissection (LCM). This process involves outlining the desired regions, which are cut on membrane slides and captured using a laser. Using LCM requires visualization and identification of the target tissue. In the case of lymphedema therapies, the target tissue is lymphatic vasculature. Rationale of Project: While lymphatics can be visualized using immunohistochemistry antibodies specific to lymphatic markers, the process is time consuming and can interfere with RNA levels in the tissue. Another option to visualize lymphatics is to use Lyve1-Cre+ mice. These mice express enhanced green fluorescent protein on lymphatic cells. The purpose of this project is to assess the utility of Lyve1-Cre+ and develop the methodology to capture the lymphatics in the murine tail to enable utilization of this methodology in murine tail model of lymphedema. Methodology Development: Samples of Lyve1-Cre+ mouse tails were harvested. Sections (10µm) were captured on LCM slides and dehydrated. The slides were visualized on the LCM microscope system, lymphatics vessels fluoresced green. They were captured via laser dissection. The captured samples were analyzed for the lymphatic specific genes (Prox1 and Lyve1) via quantitative real-time PCR to determine the purity of capture.

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213 ANALYSIS OF STEROID HORMONE RECEPTOR GENE EXPRESSION IN THE BOVINE OVARY AFTER LASER-CAPTURE MICRODISSECTION

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab213 ◽

2010 ◽

Vol 22 (1) ◽

pp. 264

Author(s):

R. Kenngott ◽

G. Palma ◽

M.J. Wendl ◽

M. Vermehren ◽

F. Sinowatz

Keyword(s):

Gene Expression ◽

Estrogen Receptor ◽

Real Time ◽

Laser Capture Microdissection ◽

Real Time Pcr ◽

Quality Indicator ◽

Estrogen Receptor Alpha ◽

Qrt Pcr ◽

Theca Interna ◽

Laser Capture

Developmental processes in complex organs like the ovary are difficult to study in terms of a biochemical and molecular biological analysis. Laser-assisted microdissection allows the efficient and precise capture of single cells or groups of cells of an organ within the context of time and space and permits their subsequent molecular characterization. Together with real-time PCR techniques, it is now feasible to study gene expression in defined cell populations of complex tissues, but it is essential to create standards optimized for fixation, preparation, and isolation of RNA, reverse transcription reaction, and real-time PCR protocol for every tissue of interest. The aim of our study was to develop protocols for a precise analysis of estrogen receptor alpha (ER-α) and progesterone receptor (PR) in defined compartments of the ovary (granulosa cells, theca interna cells, zona vasculosa, and zona parenchymatosa of the stroma). Additionally, the receptor proteins were localized by immunohistochemistry. A special focus was put on the question of how formalin fixation and paraffin embedding influences the quality of the isolated RNA from microdissected material, which was used for quantitative reverse transcription-PCR (qRT-PCR). Quality and quantity of total RNA extracted from formalin-fixed, paraffin-embedded (FFPE) sections and from material immersed in RNAlater® (Ambion, Foster City, CA, USA) was checked using an Experion automated electrophoresis system (Bio-Rad, Munich, Germany). The RNA quality indicator for microdissected material was between 6 and 7, and for RNAlater® material was 9 or better. Online qRT-PCR using the iCycler SYBR GreenTM protocol (Bio-Rad) was performed in a 96-well plate. Primer pairs were chosen to generate PCR products between 100 bp (ER-α) and 140 bp (PR), as RNA recovered from FFPE-laser microdissected material was expected to be considerably fragmented. Using GenEx software (BioEPS, Freisling, Germany), we showed that the expression of mRNA for PR was much stronger in the theca interna than in the 3 other compartments. Estrogen receptor alpha, on the other hand, was nearly exclusively expressed in the zona parenchymatosa and zona vasculosa of the stroma. Our results show that cells obtained after laser microdissection from FFPE ovarian material can be successfully used for subsequent real-time PCR, despite the fact the RNA quality indicator number of the isolated RNA was usually comparatively low. The data of our immunohistochemical analysis support the expression data of our RNA studies. In conclusion, laser-capture microdissection in combination with quantitative PCR is a reproducible and reliable technique for quantification of a small number of cells from FFPE material. We gratefully acknowledge the continuous support by the DFG-Graduiertenkolleg 1029 and the BMBF (ARG 08/013).

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Ensuring good quality rna for quantitative real-time pcr isolated from renal proximal tubular cells using laser capture microdissection

BMC Research Notes ◽

10.1186/1756-0500-7-62 ◽

2014 ◽

Vol 7 (1) ◽

Cited By ~ 13

Author(s):

Jie Yin Yee ◽

Lie Michael George Limenta ◽

Keith Rogers ◽

Susan Mary Rogers ◽

Vanessa SY Tay ◽

...

Keyword(s):

Real Time ◽

Laser Capture Microdissection ◽

Real Time Pcr ◽

Quantitative Real Time Pcr ◽

Proximal Tubular Cells ◽

Tubular Cells ◽

Proximal Tubular ◽

Renal Proximal Tubular Cells ◽

Renal Proximal Tubular ◽

Laser Capture

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Combining laser capture microdissection with quantitative real-time PCR: Effects of tissue manipulation on RNA quality and gene expression

Journal of Neuroscience Methods ◽

10.1016/j.jneumeth.2005.10.010 ◽

2006 ◽

Vol 153 (1) ◽

pp. 71-85 ◽

Cited By ~ 57

Author(s):

Ilan A. Kerman ◽

Bradley J. Buck ◽

Simon J. Evans ◽

Huda Akil ◽

Stanley J. Watson

Keyword(s):

Gene Expression ◽

Real Time ◽

Laser Capture Microdissection ◽

Real Time Pcr ◽

Quantitative Real Time Pcr ◽

Rna Quality ◽

Tissue Manipulation ◽

Laser Capture

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Somatic Mitochondrial DNA Point Mutations Used as Biomarkers to Demonstrate Genomic Heterogeneity in Primary Prostate Cancer

Prostate Cancer ◽

10.1155/2020/7673684 ◽

2020 ◽

Vol 2020 ◽

pp. 1-10

Author(s):

Christian Arstad ◽

Kristin Taskén ◽

Paulo Refinetti ◽

Ulrika Axcrona ◽

Karl-Erik Giercksky ◽

...

Keyword(s):

Prostate Cancer ◽

Mitochondrial Dna ◽

Real Time ◽

Laser Capture Microdissection ◽

Real Time Pcr ◽

Copy Number ◽

Point Mutations ◽

Primary Prostate Cancer ◽

Grade Groups ◽

Laser Capture

Primary prostate tumor heterogeneity is poorly understood, leaving research efforts with challenges regarding the initiation and advancement of the disease. The growth of tumor cells is accompanied by mutations in nuclear and in mitochondrial genomes. Thus, mitochondrial DNA mutations may be used as tumor cell markers. By the use of laser capture microdissection coupled with assays for mitochondrial point mutation detection, mtDNA mutations were used to trace mutated cells at a histological level. Point mutations in mtDNA were determined in 12 primary prostate cancers. The tumors represent different pathology-prognostic grade groups. Known mutational hotspots of the mtDNA were scanned for heteroplasmy. All specimens with mtDNA heteroplasmy were subsequently subsampled by laser capture microdissection. From a total number of 1728 microsamples, mitochondrial DNA target sequences were amplified and base substitutions detected by cycling temperature capillary electrophoresis. Real-time PCR was used as a quantitative assay to determine the relative mtDNA copy number of 12 tumors studied, represented by two samples from each (N = 24); a high degree (75%) demonstrated tumor specimen heterogeneity. A grid of 96 spots isolated by laser capture microdissection demonstrated interfocal sample heterogeneity and increased the limit of detection. The spots demonstrated a wide range of mutant fractions from 0 to 100% mutant copies. The mitochondrial DNA copy number in the samples was determined by real-time PCR. No correlation between copy number and pathology-prognostic grade groups was observed. Somatic mitochondrial DNA point mutations represent traceable biomarkers demonstrating heterogeneity in primary prostate cancer. Mutations can be detected in areas before changes in tissue histopathology are evident to the pathologist.

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