alexa Laser Capture Microdissection of Murine Interzone Cells: Layer Selection and Prediction of RNA Yield
ISSN: 2157-7633

Journal of Stem Cell Research & Therapy
Open Access

Like us on:
OMICS International organises 3000+ Global Conferenceseries Events every year across USA, Europe & Asia with support from 1000 more scientific Societies and Publishes 700+ Open Access Journals which contains over 50000 eminent personalities, reputed scientists as editorial board members.

Open Access Journals gaining more Readers and Citations

700 Journals and 15,000,000 Readers Each Journal is getting 25,000+ Readers

This Readership is 10 times more when compared to other Subscription Journals (Source: Google Analytics)

Research Article

Laser Capture Microdissection of Murine Interzone Cells: Layer Selection and Prediction of RNA Yield

Florien Jenner1*, Gerjo JVM van Osch2, Mairead Cleary2, Iris Ribitsch1, Ulrich Sauer3, René van Weeren4 and Pieter Brama5

1University of Veterinary Medicine Vienna, Equine Hospital, Vienna, Austria

2Erasmus MC, Department of Orthopaedics and Otorhinolaryngology, University Medical Center Rotterdam, Rotterdam, The Netherlands

3ZEISS Microscopy Labs, Munich, Germany

4Faculty of Veterinary Medicine, Department of Equine Sciences, Utrecht University, Utrecht, The Netherlands

5School of Veterinary Medicine, Section Veterinary Clinical Sciences, University College Dublin, Belfield, Dublin 4, Ireland

Corresponding Author:
Florien Jenner
University of Veterinary Medicine Vienna
Equine Hospital, Vienna, Austria
Tel: 431250775500
E-mail: [email protected]

Received Date: February 18, 2014; Accepted Date: March 20, 2014; Published Date: March 22, 2014

Citation: Jenner F, van Osch GJVM, Cleary M, Ribitsch I, Sauer U, et al. (2014) Laser Capture Microdissection of Murine Interzone Cells: Layer Selection and Prediction of RNA Yield. J Stem Cell Res Ther 4:183. doi:10.4172/2157-7633.1000183

Copyright: © 2014 Jenner F, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

 

Abstract

Objective: Articular chondrocytes originate from a distinct cohort of progenitor cells located in the so-called interzone in embryonic developing joints. We conducted this study 1) to determine if it is possible to identify the intermediate and outer interzone layers histologically (using cresyl violet) without adjunct in situ hybridization or immunohistochemistry; 2) to establish whether sufficient amounts of RNA can be harvested from each interzone layer of individual embryos to allow gene expression analysis; 3) to develop measurements that can provide an estimation of the RNA yield prior to costly amplification steps. Methods: Cells from the outer (OI) and intermediate (II) interzone of the nascent femorotibial joint and the epiphyseal cartilage (EC) of femur and tibia of murine embryos of 13.5 and 15.5 days gestation were harvested using laser capture microdissection (LCM). Subsequently, microarray analysis was performed to confirm appropriate layer selection. The surface area harvested and the grey value (gv) of photomicrographs taken during LCM was measured and the corresponding relative optical density (ROD) was calculated and degree and significance of the correlation with RNA yield were determined. Results: Cells from the OI, II and EC could be identified histologically using cresyl violet staining and were successfully harvested with LCM yielding sufficient amounts of RNA for linear amplification and microarray analysis. The RNA yield correlated significantly with the tissue surface area harvested, the mean gv and the corresponding ROD. Conclusions: This study provides a technique for selective laser capture microdissection and subsequent microarray analysis of murine interzone cells of the intermediate and outer layer and presents a method to estimate the RNA yield by measuring the tissue area harvested and calculating the ROD. We recommend to harvest a minimum of 1×106 μm2 of 13.5 days murine embryos and 3×106 μm2 of 15.5 days murine embryos to obtain approximately 10 ng total RNA that can be used for linear T7-based amplification and subsequent microarray analysis.

Keywords

Share This Page

Additional Info

Loading
Loading Please wait..
 
Peer Reviewed Journals
 
Make the best use of Scientific Research and information from our 700 + peer reviewed, Open Access Journals
International Conferences 2017-18
 
Meet Inspiring Speakers and Experts at our 3000+ Global Annual Meetings

Contact Us

Agri, Food, Aqua and Veterinary Science Journals

Dr. Krish

[email protected]

1-702-714-7001 Extn: 9040

Clinical and Biochemistry Journals

Datta A

[email protected]

1-702-714-7001Extn: 9037

Business & Management Journals

Ronald

[email protected]

1-702-714-7001Extn: 9042

Chemical Engineering and Chemistry Journals

Gabriel Shaw

[email protected]

1-702-714-7001 Extn: 9040

Earth & Environmental Sciences

Katie Wilson

[email protected]

1-702-714-7001Extn: 9042

Engineering Journals

James Franklin

[email protected]

1-702-714-7001Extn: 9042

General Science and Health care Journals

Andrea Jason

[email protected]

1-702-714-7001Extn: 9043

Genetics and Molecular Biology Journals

Anna Melissa

[email protected]

1-702-714-7001 Extn: 9006

Immunology & Microbiology Journals

David Gorantl

[email protected]

1-702-714-7001Extn: 9014

Informatics Journals

Stephanie Skinner

[email protected]

1-702-714-7001Extn: 9039

Material Sciences Journals

Rachle Green

[email protected]

1-702-714-7001Extn: 9039

Mathematics and Physics Journals

Jim Willison

[email protected]

1-702-714-7001 Extn: 9042

Medical Journals

Nimmi Anna

[email protected]

1-702-714-7001 Extn: 9038

Neuroscience & Psychology Journals

Nathan T

[email protected]

1-702-714-7001Extn: 9041

Pharmaceutical Sciences Journals

John Behannon

[email protected]

1-702-714-7001Extn: 9007

Social & Political Science Journals

Steve Harry

[email protected]

1-702-714-7001 Extn: 9042

 
© 2008-2017 OMICS International - Open Access Publisher. Best viewed in Mozilla Firefox | Google Chrome | Above IE 7.0 version
adwords