Fixation and Laser Capture Microdissection of Plant Tissue for RNA Extraction and RNASeq Library Preparation

2018 ◽  
Vol 3 (1) ◽  
pp. 14-32
Author(s):  
Suchitra Chavan ◽  
Elise Schnabel ◽  
Christopher Saski ◽  
Julia Frugoli
Keyword(s):  
Laser Capture Microdissection ◽  
Plant Tissue ◽  
Rna Extraction ◽  
Library Preparation ◽  
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

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.

Isolating Human Notochord Using Laser Capture Microdissection

2016 ◽  
Vol 77 (S 02) ◽  
Author(s):  
Youssef Yakkioui ◽  
Remco Santegoeds ◽  
Koo van Overbeeke ◽  
Andreas Herrler ◽  
Yasin Temel
Keyword(s):  
Laser Capture Microdissection ◽  
Laser Capture

scholarly journals Stromal MED12 exon 2 mutations in complex fibroadenomas of the breast

2020 ◽  
pp. jclinpath-2020-207062
Author(s):  
Edaise M da Silva ◽  
Francisco Beca ◽  
Ana Paula Martins Sebastiao ◽  
Melissa P Murray ◽  
Catarina Silveira ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Laser Capture Microdissection ◽  
Sanger Sequencing ◽  
Mediator Complex ◽  
Telomerase Reverse Transcriptase ◽  
Exon 2 ◽  
Stromal Component ◽  
Hotspot Mutations ◽  
Laser Capture

AimsHere we explore the presence of mediator complex subunit 12 (MED12) exon 2 and telomerase reverse transcriptase (TERT) promoter hotspot mutations in complex fibroadenomas (CFAs) of the breast.MethodsThe stromal components from 18 CFAs were subjected to Sanger sequencing of MED12 exon 2 and the TERT promoter hotspot loci. The epithelial and stromal components of two MED12 mutated CFAs were subjected to laser capture microdissection, and Sanger sequencing of MED12 exon 2, TERT promoter and PIK3CA exons 9 and 20, separately.ResultsMED12 exon 2 mutations were identified in the stroma of 17% of CFAs. The analyses of epithelial and stromal components, microdissected separately, revealed that MED12 mutations were restricted to the stroma. No TERT promoter or PIK3CA mutations in exons 9 and 20 were detected in analysed CFAs.ConclusionsLike conventional fibroadenomas, MED12 exon 2 mutations appear to be restricted to the stromal component of CFAs, supporting the notion that CFAs are stromal neoplasms.

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