JMD Association for Molecular Pathology 2008 Annual Meeting
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS

This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by von Smolinski, D.
Right arrow Articles by Gebert, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by von Smolinski, D.
Right arrow Articles by Gebert, A.
JMD 2006, Vol. 8, No. 2
Copyright © 2006 American Society for Investigative Pathology & Association for Molecular Pathology

Validation of a Novel Ultra-Short Immunolabeling Method for High-Quality mRNA Preservation in Laser Microdissection and Real-Time Reverse Transcriptase-Polymerase Chain Reaction

Dorthe von Smolinski, Maike Blessenohl, Carsten Neubauer, Kathrin Kalies and Andreas Gebert

From the Institute of Anatomy, University of Lübeck, Lübeck, Germany

Laser microdissection allows isolation of tiny samples from tissue sections for analysis of gene expression by real-time quantitative polymerase chain reaction (PCR). Although immunohistochemical labeling is often required to identify target structures, it drastically degrades mRNA so that shortened protocols are needed. Here, we present a novel method that allows fluorescence double labeling to be performed in only one incubation of 5 minutes. Fab fragments directly coupled to fluorochromes are linked to primary antibodies before these complexes are applied to sections. We quantified the influences of fixatives, labeling solutions, and incubation time on the mRNA yield and compared our method with previously proposed protocols. While tissue components, ie, vimentin and Ki67 antigen, were sufficiently stained after only 5 minutes of incubation, the new method produced a minute loss of mRNA that did not significantly differ from that of untreated sections. In contrast, incubation times of 15 and 30 minutes reduced the mRNA yield by 99.8 to 99.9%. Furthermore, incubation periods longer than 5 minutes critically affected the ratio between the target and housekeeping genes tested by factors of up to 10.6. In conclusion, the novel method described here reduces mRNA loss and potential ratio shifts to a level that does not significantly differ from that of unlabeled samples.




This article has been cited by other articles:


Home page
 GutHome page
J Buning, N Homann, D von Smolinski, F Borcherding, F Noack, M Stolte, M Kohl, H Lehnert, and D Ludwig
Helminths as governors of inflammatory bowel disease
Gut, August 1, 2008; 57(8): 1182 - 1183.
[Full Text] [PDF]

Home page
 Biophys. JHome page
A. Vogel, K. Lorenz, V. Horneffer, G. Huttmann, D. von Smolinski, and A. Gebert
Mechanisms of Laser-Induced Dissection and Transport of Histologic Specimens
Biophys. J., December 15, 2007; 93(12): 4481 - 4500.
[Abstract] [Full Text] [PDF]


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Copyright © 2006 by the American Society for Investigative Pathology and the Association for Molecular Pathology.