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 -

Use of Degenerate PCR

[PDF]

Use of Degenerate PCR to Identify an Anti-apoptotic Gene of BHV-1 ...

We have undertaken the identification of a BHV-1 anti-apoptotic gene homologous. to c34.5 of HSV-1, using the degenerate PCR method. ... www.springerlink.com/index/X9012U4016U47J07.pdf -

Degenerate and Nested PCR

Degenerate and Nested PCR: a Highly Sensitive and Specific Method ...

In summary, we have established a highly sensitive and comprehensive degenerate PCR methodology for detection and genotyping of HPV from the skin and have ... jcm.asm.org/cgi/content/abstract/37/11/3545

Degenerate PCR and its application in gene cloning

[Degenerate PCR and its application in gene cloning]

Degenerate PCR is introduced in this paper, including what is degenerate PCR, how to design degenerate primers, how to optimize degenerate PCR parameters, ... www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=PubMed&list_uids=15639854&dopt=AbstractPlus -

Degenerate PCR

Degenerate PCR

The identification of novel members of gene families by PCR using degenerate primers is described and protocols given. Article by Michael Koelle 1996 on the ... www.dartmouth.edu/~ambros/protocols/other/koelle/degenerate_PCR.html - 17k - Cached -

Degenerate PCR

Degenerate PCR

An introduction with principle, applications and description of the technique. From the web site of Kebangsaan University, Malaysia. cgat.ukm.my/protease/degpcr.html - 11k - Cached -

Keunhwa's Inverse PCR Protocol

[DOC]

Keunhwa's Inverse PCR Protocol

File Format: Microsoft Word - View as HTML Keunhwa's Inverse PCR Protocol. 1. Extract plant genomic DNA using the Qiagen DNeasy kit. Elute in 50㎕ EB buffer. 2. Digest 15㎕ DNA sample (~ 300ng) with ... bio.kaist.ac.kr/~pdbl/Inverse.doc -

Inverse PCR-Based RFLP Scanning

[PDF]

Inverse PCR-Based RFLP Scanning Identifies Low-Level Mutation ...

We describe inverse PCR-based, amplified restriction fragment ... high-throughput inverse PCR in conjunction with automated denatur- ... cancerres.aacrjournals.org/cgi/reprint/64/7/2544.pdf -