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In Situ PCR A Review

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In Situ PCR A Review

[Frontiers in Bioscience 2, c15-29, September 1, 1997]

    IN SITU PCR. OVERVIEW OF PROCEDURES AND APPLICATIONS

     Carlos A. Muro-Cacho, M.D., Ph.D.

Department of Pathology, H. Lee Moffitt Cancer Center and Research Institute and University of South Florida College of

    Medicine, Tampa, Florida, USA

TABLE OF CONTENTS

     1. Abstract

     2. Introduction

     3. General considerations

     3.1. Equipment

     3.2 Technical aspects

     3.3 Types of samples

     3.4 Fixation

     3.5 Proteolytic digestion

     3.6 Additional pretreatments

     4. Polymerase Chain Reaction (PCR)

    4.1 Polymerases

    4.2 Deoxynucleotide triphosphates (dNTPs)

    4.3 Buffers

    4.4 Primers

    4.5 Cycling profile

     5. In situ PCR Protocols

    5.1 In situ RT-PCR

    5.2 Direct In situ PCR

    5.3 Indirect In situ PCR

     6. Controls

    6.1 Causes of false negativity

    6.2 Causes of false positivity

    6.3 DNA repair

    6.4 Mispriming

    6.5 Endogenous priming

    6.6 Diffusion of PCR products

     7. Applications

     8. Acknowledgment

     9. References

     10. Appendices

     Appendix 1 - General information

     Appendix 2 - Sample preparation Appendix 3 - Proteolytic digestion Appendix 4 - Additional pretreatments Appendix 5 - RT-PCR Appendix 6 - Polymerase Chain Reaction Appendix 7 - Direct In situ PCR Appendix 8 - Indirect In situ PCR Appendix 9 - Detection methods morphology is essential to the full understanding of cell 1. ABSTRACT biology. A variety of methods for detection of nucleic acids are currently available. Solution PCR requires disruption of The evaluation of gene expression in the context of cellular the sample and detection of the amplified material by _____________________________________________________ electrophoresis in agarose gels. In situ Hybridization Received 1/10/97 Accepted 7/15/97 methods, on the other hand, permit morphological correlation Send correspondence to: Carlos A. Muro-Cacho, M.D., Ph.D. and provide a high sensitivity that is sufficient for many Pathology Department, H.Lee Moffitt Cancer Center and applications. In some instances, however, the amount of Research Institute at University of South Florida, College of target in the sample is below the limit of detection of this Medicine, 12902 Magnolia Drive, Tampa, FL 33612-9497 technique. In situ PCR allows the detection of minimal Tel: (813) 972-4673 ext. 2268, FAX:(813) 632-1708, E-mail: amounts of nucleic acids with exquisite sensitivity and murocacho@moffitt.usf.edu

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In Situ PCR; An Overview

    specificity, while the integrity of the cells and the morphology procedures that would be otherwise restricted to highly of tissues remains preserved. This technique, although not specialized basic science research groups. The adaptation of exempt from difficulties, is undergoing methodological some of these procedures, to the detection of nucleic acids in simplifications that will make it suitable for an increasing cells and tissues, converts genetic information into a visual number of basic science and clinical applications. The signal that can be evaluated In situ while preserving cellular

    following is a review of the principles, methods and integrity and tissue morphology (1-16). This signal can then applications of In situ PCR. be evaluated in the context of a homogeneous or

     heterogeneous cell population, a given cellular compartment, 2. INTRODUCTION or a particular pathological tissue change. In situ PCR is also

     known as PCR In situ (1), PCR ISH (2), In-cell PCR (3), and

    The last decade has witnessed a continuous PCR-driven ISH (4), and for those modifications intended to revolution in molecular biology methods and the parallel amplify mRNA, RT In situ PCR (5) or In situ cDNA PCR (6) development of a competitive biotechnology industry that has (appendix 1 and figure 1). made available high quality reagents and sophisticated equipments at a progressively lower cost. This has allowed the routine use, by many laboratories, of state-of-the-art

     SAMPLE PREPARATION

     FIXATION

     PARAFFIN EMBEDDING

     SECTIONING AND SLIDE PREPARATION

     DEPARAFFINATION

     REHYDRATION

     PROTEOLYTIC DIGESTION

     IN SITU PCR IN SITU RT-PCR

     DNase DIGESTION (optional)

     RT

     DNA AMPLIFICATION

     DIRECT INDIRECT

     IHC ISH

     VISUALIZATION

    Figure 1. In situ PCR protocols for tissue sections

Abbreviations: PCR = Polymerase Chain Reaction, RT = Reverse Transcription, ISH = In situ Hybridization, IHC =

    Immunohistochemistry

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In Situ PCR; An Overview

    polish application and removal, transfers in and out of the 3. GENERAL CONSIDERATIONS

    thermocycler, etc.).

     3.1 Equipment

     Thermocyclers are designed to reach and maintain 3.3 Types of samples (appendix 2)

    the necessary temperatures in a reproducible manner over In situ PCR has been successfully performed on a specified periods of time and predetermined numbers of variety of samples: cell smears, imprints, cell blocks, cycles. Some of these instruments have independent "slide" cytospins, metaphase chromosomes, frozen sections and blocks and "tube" blocks, connected to a common sections of tissues embedded in plastic or paraffin. Haase et programmable controlling unit, providing the opportunity to al (14) performed In situ PCR on fixed cells suspended in the run different protocols simultaneously and to correlate PCR reaction mixture within a microcentrifuge tube. In this solution-PCR with In situ PCR results. The commercially procedure, after amplification, cells are recovered by available thermocyclers, use different mechanisms to prevent cytocentrifugation onto glass slides (3, 4, 8, 12, 14). An evaporation and increase efficiency and consistency of results. advantage of this method is that cells may be lysed after In the Omnislide Temperature Cycling System, Hybaid amplification and the lysate analyzed by gel electrophoresis (Teddington, Middlesex, UK, http://www.hybaid.co.uk/), and Southern blot hybridization (14, 15). Good results can slides are placed in a sealed humid chamber. In the PTC-also be obtained in cytospins and cell blocks prepared 100-12MS Programmable Thermal Controller (MJ Research, following standard protocols. When tissues are used, 4-6 Inc., Watertown, MA, USA, http://www.mjr.com/), the micron thick sections are prepared from paraffin or frozen reagents are added directly to the amplification mixture to tissue blocks. The morphological changes induced by avoid evaporation. With the Gene-Amp In situ PCR System freezing, and the increase rate of diffusion of the amplified 1000 (Perkin-Elmer Corp., Norwalk, CT, USA, product, make frozen sections less desirable for In situ PCR

    purposes. Although thicker sections may compromise http://www.perkin-elmer.com/), up to 3 samples/slide can

    evaluation of morphology and increase noise to signal ratio, it be tested by clamping slides individually using water-tight

    is believed that thick paraffin sections contain more starting seals. Several cheaper ovens, in which In situ PCR can be nucleic acid material and allow a better yield of amplified performed, are also available. The BioOven from Biotherm signal. Deparaffinization is performed on sections cut from Corp. (Fairfax, VA, USA), ensures temperature control via a paraffin blocks, according to standard procedures by sensor placed under the coverslip or glass slide that sends removing paraffin in xylene and rehydration in decreasing information to the control unit. Those that already have a concentrations of ethanol. conventional solution PCR thermocycler can also perform In situ PCR experiments by placing slides in a "tube" block. In 3.4 Fixation (appendix 2) these systems, however, heat transfer to the tissues is less The goal of fixation is to maintain optimal tissue reliable. morphology while preserving the integrity of the nucleic acids. It is generally agreed that aldehyde-based fixatives, such as 3.2 Technical aspects 10% neutral buffered formalin, are superior for preservation The same precautions that apply to solution PCR of morphology and for minimizing the diffusion of signal (17-procedures, should be exercised in In situ PCR experiments. 20). This is fortunate since the vast majority of archival Due to the extreme amplification capability of the In situ PCR, material in pathology departments has been fixed in 10% any small amount of contaminant DNA in the sample can be neutral buffered-formalin. Nevertheless, not all routine inadvertently amplified. During specimen handling, areas procedures for fixation of specimens are done under ideal where amplified DNA (in particular previously amplified standard conditions and, for most protocols, a variety of DNA) can be found should be avoided. Devices (positive proteolytic digestion conditions has to be tested. This is due, displacement pipets, pipet tips containing sterile filters, tubes, in part, to cross-linkages between proteins, or between etc.) have to be specifically allocated to In situ PCR histones and DNA, which are difficult to remove and may experiments and frequent changes of gloves have to become impair the efficiency of PCR. Furthermore, aldehyde-based routine. When working with tissue sections, appropriately fixatives may induce single-stranded breaks that, when coated slides (i.e., poly-L-lysine, Aminosilane) should be used repaired by the DNA polymerase, result in false positivity. to avoid the detachment of sections during the proteolytic Fixatives containing picric acid (Bouin's solution) or mercury digestion step (16-17). To avoid evaporation, a variety of (Zenker's solution) seem to degrade nucleic acids and methods have been attempted. The section is overlaid with generally are not recommended (17-22). Tissues, preferably the PCR mixture and a coverslip is placed over the section. less than 0.5 cm in thickness, are fixed at room temperature The coverslip may be sealed with nail polish or rubber cement in 10 % Neutral Buffered Formalin. Most cell suspensions making sure not to introduce bubbles. After amplification, and cytology preparations are routinely fixed in 95 % alcohol the seal is peeled-off with forceps and the coverslip removed o for 1 hour either at 4C or at room temperature. They can be in 0.1X SSC. Alternatively, specially designed cones (1, 8) o washed in PBS and kept at 4C for short-term storage or are applied to the slide sealing the area of interest. The o dehydrated and placed at 70C for long-term storage. amplification solution is added to the cones making sure that Paraformaldehyde also provides excellent results, being the it diffuses evenly over the tissue section without creating air fixative of choice in many laboratories (21, 22). It can be bubbles. If the operator does not have previous experience used at concentrations ranging from 1 % to 4 % during a with the procedure, it is advisable to attempt several dry runs o period of 4 to 24 hours, preferably at 4C. Fixation has to to get acquainted with the peculiarities of the technique (slide be initiated as early as possible to reduce the chance of manipulation, microscope checks, rubber cement and nail