Reverse Transcriptase In Situ PCR Technique

Reverse Transcriptase In Situ PCR Technique Source CSH Protocols; 2006; doi:10.1101/pdb.prot4119 Overview This protocol describes a method for reverse transcriptase (RT) in situ PCR. In situ PCR differs from PCR in situ hybridization in the inclusion of a reporter molecule in the amplification step. The two steps of RT in situ PCR that differ from in situ PCR are overnight digestion in RNase-free DNase that is performed after protease digestion, and an RT step, prior to in situ PCR. By Gerard J. Nuovo.

http://www.biocompare.com/jumpres.asp?type=protocol&protocolid=2441&siteid=30

In situ PCR

In situ PCR for detection and identification of fungal species

To overcome this problem, we designed an in situ PCR technique that links PCR ... first report of in situ PCR on phytopathogenic fungal material. ... journals.cambridge.org/production/action/cjoGetFulltext?fulltextid=110878

In situ PCR Expression Analysis

FGP: in situ PCR Expression Analysis

Method: in situ PCR expression analysis (method details to be added). There have been 10374 hits since 09/26/2003. Please note: suggested browsers are ... www.floralgenome.org/fgp/methods/method_in_situ.html - 10k - Cached

PCR In Situ Hybridization: Protocols and Applications

Archives of Pathology & Laboratory Medicine,  Jul 1997  by Hinrichs, Steven H

2nd ed, by Gerald Nuovo, 449 pp, with illus, $80, New York, NY, Raven Press, 1994.

This book represents a renewed effort to bring procedures in molecular biology into the domain of anatomic pathology research. For pathologists who cut their teeth on avidin-biotin immunohistochemistry and only recently became comfortable with polymerase chain reaction (PCR) techniques, this book is a logical entry point into leading edge molecular diagnostics and research using PCR in situ hybridization...

http://findarticles.com/p/articles/mi_qa3725/is_199707/ai_n8764476

Nested in situ PCR-ISH method

Development of a novel nested in situ PCR-ISH method for detection of hepatitis C virus RNA in liver tissue

Authors: Alzahrani A.J.; Vallely P.J.; McMahon R.F.T.¹

Source: Journal of Virological Methods, Volume 99, Number 1, January 2002 , pp. 53-61(9)

Publisher: Elsevier

http://www.ingentaconnect.com/content/els/01660934/2002/00000099/00000001/art00383

Co-labeling Using In Situ PCR

Journal of Histochemistry and Cytochemistry, Vol. 49, 1329-1340, November 2001, Copyright © 2001, The Histochemical Society, Inc.

REVIEW

Co-labeling Using In Situ PCR: A Review

Gerard J. Nuovo^a
a Department of Pathology, Ohio State University Medical Center, Columbus, Ohio

Correspondence to: Gerard J. Nuovo, Dept. of Pathology, Ohio State U. Medical Center, S305 Rhodes Hall, 450 W 10th Ave., Columbus, OH 43210. E-mail: gnuovomd@pol.net

Summary

In situ amplification permits the histological localization of low-copy DNA and RNA targets. However, in many instances it would be useful to know the specific phenotype of the target-containing cell or to ascertain the distribution of a different nucleic acid sequence in the same tissue section. This review describes a methodology that allows co-in situ localization of two nucleic acid targets or a DNA/RNA sequence and a protein in paraffin-embedded, formalin-fixed tissue. The key variable for detection of low-copy RNA targets by RT in situ PCR is optimal protease digestion to permit cDNA target-specific incorporation of the reporter nucleotide. This is achieved via inactivation of nonspecific DNA synthesis by overnight DNase digestion. The key variable for immunohistochemical localization of proteins is to determine the effect of protease digestion on the antigen-based signal intensity. Background for DNA targets by in situ hybridization or, for targets present in 1–10 copies per cell, PCR ISH is dependent primarily on probe concentration and the stringency of the post-hybridization wash. Radioactive ³H-labeled nucleotides permit an excellent distinction with colorimetric signals for co-localization, although two distinct chromogens can in many instances allow successful localization of two different targets.

http://www.jhc.org/cgi/content/full/49/11/1329

In situ reverse trancriptase-polymerase chain reaction

J Nanobiotechnology. 2003; 1: 3.

Published online 2003 May 28. doi: 10.1186/1477-3155-1-3.

 

Copyright © 2003 Stamps et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.

 

Application of in situ reverse trancriptase-polymerase chain reaction (RT-PCR) to tissue microarrays

Alasdair C Stamps,¹ Jonathan A Terrett,¹ and Paul J Adam¹

¹Oxford GlycoSciences (UK) Ltd., The Form, 86 Milton Park, Abingdon, UK OX14 4RY

Corresponding author.

Alasdair C Stamps: astamps@ntlworld.com ; Jonathan A Terrett: jon.terrett@ogs.co.uk ; Paul J Adam: paul.adam@ogs.co.uk

 

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=161785

 

 

RT IN SITU PCR

THE FOUNDATION OF SUCCESSFUL RT IN SITU PCR

Gerard J. Nuovo

Director, MGN Medical Research Laboratories, Setauket, New York 11733, USA

Received: 07/25/96; Accepted: 10/02/96; On-line 11/01/96

TABLE OF CONTENTS

1. Abstract

2. Introductory statement

3. The key preparatory steps

3.1. Fixative

3.1.1. Protease digestion

3.1.2. Definition of optimal protease digestion

3.1.3. Definitions of suboptimal and over-digestion

3.1.4. DNase digestion

3.1.5. Direct incorporation of the reporter nucleotide

4. A one step protocol for RT in situ PCR

5. Applications of RT in situ PCR Matrix metalloprotease expression in cancer as a model for RT in situ PCR

5.1. MMP and TIMP expression in cervical cancer

6. Concluding remarks

7. References

8. Entire manuscript

Key words: In situ, PCR, reverse transcriptase

http://www.bioscience.org/1996/v1/c/nuovo1/htmls/list.htm

In Situ PCR

Definition of In Situ PCR

In Situ PCR (ISH) is a polymerase chain reaction that actually takes place inside the cell on a slide. In situ PCR amplification can be performed on fixed tissue or cells.

Full Details Here

http://www.pcrstation.com/in-situ-pcr/ 

 

PCR IN SITU

PCR IN SITU: NEW TECHNOLOGIES WITH SINGLE CELL RESOLUTION FOR THE DETECTION AND INVESTIGATION OF VIRAL LATENCY AND PERSISTENCE

Katherine A. Staskus, Janet E. Embretson, Ernest F. Retzel,

Janet Beneke and Ashley T. Haase

Copyright � 1994 The Regents of the University of Minnesota

Table of Contents

I. INTRODUCTION AND BACKGROUND
II. THE LENTIVIRUSES AS AN EXPERIMENTAL PROTOTYPE
III. METHODOLOGY
A. Preparation of Cells and Tissues
1. The Balance of Fixation and Pretreatment
B. Primer design
C. Conditions of Amplification
1. Cells in Suspension
2. Cells Within Tissue Sections
D. Detection of Amplified Sequences
IV. APPLICATION AND RELEVANCE TO HUMAN VIRAL DIAGNOSIS AND RESEARCH OF VIRAL PATHOGENESIS
ACKNOWLEDGMENTS
REFERENCES

In situ PCR Plant Material

These pages are dedicated to in situ PCR and related techniques on plant material. Besides a complete protocols for performing in situ PCR on plant meterial, these pages also includes references to some major papers and books on the subject, a list of in situ PCR links on the internet, and in the future a lot of tips & tricks on 'how to do'  - but this all depends on you readers.

Required materials
Required solutions
Tissue preparation
Pectinase treatment
Protease treatment
Acetylation
DNase treatment
Reverse transcription
Thermal cycling
Detection
Mounting

In Situ PCR

The In Situ PCR:

Amplification and Detection in a Cellular Context

Ernest F. Retzel, Katherine A. Staskus, Janet E. Embretson and Ashley T. Haase

Department of Microbiology, University of Minnesota, Minneapolis, MN 55455

 

Introduction
Technical Aspects/Philosophy
Primer Design
Reaction Conditions
"Mechanics" of the Reaction
Methodology
A. Cell and Tissues.
1. Cultured Cells: Infection and Preparation.
2. Tissues: Pulmonary infection and preparation.
B. In Situ Amplification
1. Cultured Cells in Suspension.
2. Tissues on Slides.
C. Detection of Amplified DNA by in situ Hybridization
1. Solution amplification.
2. Tissue Sections.
References

Gene cloning, biochemical characterization and substrate specificity

A new nitrilase from Bradyrhizobium japonicum USDA 110. Gene cloning, biochemical characterization and substrate specificity
Journal of Biotechnology, In Press, Accepted Manuscript, Available online 10 October 2007
Dunming Zhu, Chandrani Mukherjee, Yan Yang, Beatriz E. Rios, D. Travis Gallagher, N. Natasha Smith, Edward R. Biehl and Ling Hua

Site-directed mutagenesis investigation

Site-directed mutagenesis investigation of coupling properties of metal ion transport by DCT1
Biochimica et Biophysica Acta (BBA) - Biomembranes, In Press, Accepted Manuscript, Available online 16 October 2007
Yaniv Nevo

Design of a degenerate PCR primer pair to amplify signature

[DOC]

(1) Design of a degenerate PCR primer pair to amplify signature ...

File Format: Microsoft Word - View as HTML (1) Design of a degenerate PCR primer pair to amplify signature sequences for all vertebrate sarcomeric myosin heavy chain genes : Recognition of the MYH 16 ... www.nature.com/nature/journal/v428/n6981/extref/nature02358-s1.doc -