scholarly journals Laser capture microdissection of intestinal tissue from sea bass larvae using an optimized RNA integrity assay and validated reference genes

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
M. Schaeck ◽  
W. De Spiegelaere ◽  
J. De Craene ◽  
W. Van den Broeck ◽  
B. De Spiegeleer ◽  
...  
Keyword(s):  
Laser Capture Microdissection ◽  
Reference Genes ◽  
Sea Bass ◽  
Intestinal Tissue ◽  
Rna Integrity ◽  
Sea Bass Larvae ◽  
Laser Capture

scholarly journals Maintaining RNA integrity in a homogeneous population of mammary epithelial cells isolated by Laser Capture Microdissection

2010 ◽  
Vol 11 (1) ◽  
Author(s):  
Claudia Bevilacqua ◽  
Samira Makhzami ◽  
Jean-Christophe Helbling ◽  
Pierre Defrenaix ◽  
Patrice Martin
Keyword(s):  
Epithelial Cells ◽  
Laser Capture Microdissection ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Homogeneous Population ◽  
Rna Integrity ◽  
Laser Capture

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 Laser Capture Microdissection optimization for high-quality RNA in mouse brain tissue

2021 ◽  
Author(s):  
Margareth Nogueira ◽  
Daiane CF Golbert ◽  
Richardson Leão
Keyword(s):  
Brain Tissue ◽  
Mouse Brain ◽  
Laser Capture Microdissection ◽  
Basolateral Amygdala ◽  
Single Cells ◽  
Heterogeneous Material ◽  
Anterior Cingulate ◽  
High Quality ◽  
Rna Integrity ◽  
Laser Capture

Laser Capture Microdissection (LCM) is a method that allows to select and dissecting specific structures, cell populations, or even single cells from different types of tissue to extract DNA, RNA, or proteins. It is easy to perform and precise, avoiding unwanted signals from irrelevant cells, because gene expression may be affected by a bulk of heterogeneous material in a sample. However, despite its efficiency, several steps can affect the sample RNA integrity. In comparison to DNA, RNA is a much more unstable molecule and represents a challenge in the LCM method. Here we describe an optimized protocol to provide good concentration and high-quality RNA in specific structures, such as Dentate Gyrus and CA1 in the hippocampus, basolateral amygdala and anterior cingulate cortex of mouse brain tissue.

A robust RNA integrity-preserving staining protocol for laser capture microdissection of endometrial cancer tissue

2011 ◽  
Vol 416 (1) ◽  
pp. 123-125 ◽  
Author(s):  
Michele Cummings ◽  
Ciara V. McGinley ◽  
Nafisa Wilkinson ◽  
Sarah L. Field ◽  
Sean R. Duffy ◽  
...  
Keyword(s):  
Endometrial Cancer ◽  
Laser Capture Microdissection ◽  
Cancer Tissue ◽  
Rna Integrity ◽  
Staining Protocol ◽  
Laser Capture

A Systematic Comparison of Protocols for Recovery of High-Quality RNA from Human Islets Extracted by Laser Capture Microdissection v1

2021 ◽  
Author(s):  
Chiara M. A. Cefalo ◽  
Teresa Mezza ◽  
Andrea Giaccari ◽  
Rohit N. Kulkarni
Keyword(s):  
Laser Capture Microdissection ◽  
Rna Extraction ◽  
Human Islets ◽  
Human Pancreas ◽  
Sequencing Analysis ◽  
High Quality ◽  
Partial Pancreatectomy ◽  
Rna Integrity ◽  
Laser Capture ◽  
Yield And Purity

The isolation of high-quality RNA from endocrine pancreas sections represents a considerable challenge largely due to the high ribonuclease levels. Laser capture microdissection (LCM) of mammalian islets, in association with RNA extraction protocols, has emerged as a feasible approach to characterizing their genetic and proteomic profiles. However, a validated protocol to obtain highquality RNA from LCM-derived human pancreas specimens that is appropriate for next-generation sequencing analysis is still lacking. In this study, we applied four methods (Picopure extraction kit, Qiazol protocol, Qiazol + Clean-up kit, and RNeasy Microkit + Carrier) to extract RNA from human islets obtained from both non-diabetic individuals and patients with type 2 diabetes who had undergone partial pancreatectomy, as well as handpicked islets from both non-diabetic and diabetic organ donors. The yield and purity of total RNA were determined by 260/280 absorbance using Nanodrop 100 and the RNA integrity number with a bioanalyzer. The results indicated that among the four methods, the RNeasy MicroKit + Carrier (Qiagen) provides the highest yield and purity.

scholarly journals A Systematic Comparison of Protocols for Recovery of High-Quality RNA from Human Islets Extracted by Laser Capture Microdissection

Biomolecules ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 625
Author(s):  
Chiara M. A. Cefalo ◽  
Teresa Mezza ◽  
Andrea Giaccari ◽  
Rohit N. Kulkarni
Keyword(s):  
Laser Capture Microdissection ◽  
Rna Extraction ◽  
Human Islets ◽  
Human Pancreas ◽  
Sequencing Analysis ◽  
High Quality ◽  
Partial Pancreatectomy ◽  
Rna Integrity ◽  
Laser Capture ◽  
Yield And Purity

The isolation of high-quality RNA from endocrine pancreas sections represents a considerable challenge largely due to the high ribonuclease levels. Laser capture microdissection (LCM) of mammalian islets, in association with RNA extraction protocols, has emerged as a feasible approach to characterizing their genetic and proteomic profiles. However, a validated protocol to obtain high-quality RNA from LCM-derived human pancreas specimens that is appropriate for next-generation sequencing analysis is still lacking. In this study, we applied four methods (Picopure extraction kit, Qiazol protocol, Qiazol + Clean-up kit, and RNeasy Microkit + Carrier) to extract RNA from human islets obtained from both non-diabetic individuals and patients with type 2 diabetes who had undergone partial pancreatectomy, as well as handpicked islets from both non-diabetic and diabetic organ donors. The yield and purity of total RNA were determined by 260/280 absorbance using Nanodrop 100 and the RNA integrity number with a bioanalyzer. The results indicated that among the four methods, the RNeasy MicroKit + Carrier (Qiagen) provides the highest yield and purity.

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