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.

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 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.

scholarly journals Gene Expression of Purified β-Cell Tissue Obtained from Human Pancreas with Laser Capture Microdissection

2008 ◽  
Vol 93 (3) ◽  
pp. 1046-1053 ◽  
Author(s):  
Lorella Marselli ◽  
Jeffrey Thorne ◽  
Yu-Bae Ahn ◽  
Abdulkadir Omer ◽  
Dennis C. Sgroi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Laser Capture Microdissection ◽  
Human Pancreas ◽  
Β Cell ◽  
Cell Tissue ◽  
Laser Capture

Application of Novel Laser Capture Microdissection and RT-PCR to a Functional Analysis of Osteopontin in Mouse Growth Plate Cartilage

2001 ◽  
Vol 7 (S2) ◽  
pp. 44-45
Author(s):  
R. Jacquet ◽  
J. Hillyer ◽  
J. Zhang ◽  
W. J. Landis
Keyword(s):  
Growth Plate ◽  
Laser Capture Microdissection ◽  
Critical Role ◽  
Rna Extraction ◽  
Normal Growth ◽  
Alternative Possibilities ◽  
Rt Pcr ◽  
Hypertrophic Growth ◽  
Growth Plate Cartilage ◽  
Laser Capture

The long bones of vertebrates, such as the tibiae and femurs of humans, extend in length by means of genotypic and phenotypic changes orchestrated by the chondrocytes comprising growth plate cartilage. Among the constituents synthesized by these cartilage cells, osteopontin (OPN), a phosphorylated glycoprotein, is thought to play a critical role in events leading to normal growth plate function and ultimate mineralization of the deeper zones of this cartilage region. The precise role of OPN, however, is uncertain with regard to mineralization, and present evidence supports the alternative possibilities that the protein may be either facilitative or inhibitory to mineral deposition. in order to investigate OPN function in a model growth plate, cartilage from normal 1-11 day old postnatal mice was examined by the novel techique of laser capture microdissection (LCM) followed by RT-PCR to obtain a measure of OPN gene expression by chondrocytes of known age and specific location in the plate. LCM permits identification of individual or clusters of cells within a tissue section and subsequent unique isolation (“capture”) of such cells for a variety of molecular analyses.In this study, mouse tibiae were dissected, placed in RNAlater (Ambion, Austin, TX) to preserve message, and stored at −20°C. Sections (5 μm thick) of fresh frozen developing epiphyseal growth plates were obtained in a cryostat maintained at −20°C, fixed briefly in 70% ethanol, and stained with eosin. Sections were examined in a Pixcell LCM system (Arcturus Engineering, Mountain View, CA) where chondrocytes were attached to the surface of polymer film substrates and lifted free of sections. in separate experiments, ∼200-1200 cells were captured and analyzed. Substrates were transferred to Eppendorf tubes containing RNA extraction buffer. RNA was extracted from cells by microisolation (Stratagene, La Jolla, CA), DNAse-treated, reverse-transcribed, and then subjected to PCR (40 cycles) with AmpliTaq DNA polymerase (PE Applied Biosystems, Foster City, CA). Ethidium bromide agarose gels revealed OPN mRNA from groups of chondrocytes isolated from whole cartilage and from resting, proliferating, and hypertrophic growth plate zones from the mouse tibiae. Brain cells captured by LCM from the same mouse sections served as positive controls and reactions containing no reverse transcriptase were negative controls. 18S rRNA was used as a marker for semiquantitation and standardization of expressed message from captured cells.

scholarly journals Tissue preparation for laser capture microdissection and RNA extraction from fresh frozen breast tissue

BioTechniques ◽  
2007 ◽  
Vol 43 (1) ◽  
pp. 41-48 ◽  
Author(s):  
Mary Morrogh ◽  
Narciso Olvera ◽  
Faina Bogomolniy ◽  
Patrick I. Borgen ◽  
Tari A. King
Keyword(s):  
Laser Capture Microdissection ◽  
Breast Tissue ◽  
Rna Extraction ◽  
Tissue Preparation ◽  
Fresh Frozen ◽  
Laser Capture

scholarly journals High-Quality RNA from Cells Isolated by Laser Capture Microdissection

BioTechniques ◽  
2002 ◽  
Vol 33 (1) ◽  
pp. 176-179 ◽  
Author(s):  
A. Mikulowska-Mennis ◽  
T.B. Taylor ◽  
P. Vishnu ◽  
S.A. Michie ◽  
R. Raja ◽  
...  
Keyword(s):  
Laser Capture Microdissection ◽  
High Quality ◽  
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.

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