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

	[Frontiers in Bioscience 2, c15-29, September 1, 1997 Reprints PubMed CAVEAT LECTOR
	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 Received 1/10/97 Accepted 7/15/97 3. GENERAL CONSIDERATIONS 3.1 Equipment Thermocyclers are designed to reach and maintain the necessary temperatures in a reproducible manner over specified periods of time and predetermined numbers of cycles. Some of these instruments have independent "slide" blocks and "tube" blocks, connected to a common programmable controlling unit, providing the opportunity to run different protocols simultaneously and to correlate solution-PCR with In situ PCR results. The commercially available thermocyclers, use different mechanisms to prevent evaporation and increase efficiency and consistency of results. In the Omnislide Temperature Cycling System, Hybaid (Teddington, Middlesex, UK, http://www.hybaid.co.uk/), slides are placed in a sealed humid chamber. In the PTC-100-12MS Programmable Thermal Controller (MJ Research, Inc., Watertown, MA, USA, http://www.mjr.com/), the reagents are added directly to the amplification mixture to avoid evaporation. With the Gene-Amp In situ PCR System 1000 (Perkin-Elmer Corp., Norwalk, CT, USA, http://www.perkin-elmer.com/), up to 3 samples/slide can be tested by clamping slides individually using water-tight seals. Several cheaper ovens, in which In situ PCR can be performed, are also available. The BioOven from Biotherm Corp. (Fairfax, VA, USA), ensures temperature control via a sensor placed under the coverslip or glass slide that sends information to the control unit. Those that already have a conventional solution PCR thermocycler can also perform In situ PCR experiments by placing slides in a "tube" block. In these systems, however, heat transfer to the tissues is less reliable. 3.2 Technical aspects The same precautions that apply to solution PCR procedures, should be exercised in In situ PCR experiments. Due to the extreme amplification capability of the In situ PCR, any small amount of contaminant DNA in the sample can be inadvertently amplified. During specimen handling, areas where amplified DNA (in particular previously amplified DNA) can be found should be avoided. Devices (positive displacement pipets, pipet tips containing sterile filters, tubes, etc.) have to be specifically allocated to In situ PCR experiments and frequent changes of gloves have to become routine. When working with tissue sections, appropriately coated slides (i.e., poly-L-lysine, Aminosilane) should be used to avoid the detachment of sections during the proteolytic digestion step (16-17). To avoid evaporation, a variety of methods have been attempted. The section is overlaid with the PCR mixture and a coverslip is placed over the section. The coverslip may be sealed with nail polish or rubber cement making sure not to introduce bubbles. After amplification, the seal is peeled-off with forceps and the coverslip removed in 0.1X SSC. Alternatively, specially designed cones (1, 8) are applied to the slide sealing the area of interest. The amplification solution is added to the cones making sure that it diffuses evenly over the tissue section without creating air bubbles. If the operator does not have previous experience with the procedure, it is advisable to attempt several dry runs to get acquainted with the peculiarities of the technique (slide manipulation, microscope checks, rubber cement and nail polish application and removal, transfers in and out of the thermocycler, etc.). 3.3 Types of samples (appendix 2) In situ PCR has been successfully performed on a variety of samples: cell smears, imprints, cell blocks, cytospins, metaphase chromosomes, frozen sections and sections of tissues embedded in plastic or paraffin. Haase et al (14) performed In situ PCR on fixed cells suspended in the PCR reaction mixture within a microcentrifuge tube. In this procedure, after amplification, cells are recovered by cytocentrifugation onto glass slides (3, 4, 8, 12, 14). An advantage of this method is that cells may be lysed after amplification and the lysate analyzed by gel electrophoresis and Southern blot hybridization (14, 15). Good results can also be obtained in cytospins and cell blocks prepared following standard protocols. When tissues are used, 4-6 micron thick sections are prepared from paraffin or frozen tissue blocks. The morphological changes induced by freezing, and the increase rate of diffusion of the amplified product, make frozen sections less desirable for In situ PCR purposes. Although thicker sections may compromise evaluation of morphology and increase noise to signal ratio, it is believed that thick paraffin sections contain more starting nucleic acid material and allow a better yield of amplified signal. Deparaffinization is performed on sections cut from paraffin blocks, according to standard procedures by removing paraffin in xylene and rehydration in decreasing concentrations of ethanol. 3.4 Fixation (appendix 2) The goal of fixation is to maintain optimal tissue morphology while preserving the integrity of the nucleic acids. It is generally agreed that aldehyde-based fixatives, such as 10% neutral buffered formalin, are superior for preservation of morphology and for minimizing the diffusion of signal (17-20). This is fortunate since the vast majority of archival material in pathology departments has been fixed in 10% neutral buffered-formalin. Nevertheless, not all routine procedures for fixation of specimens are done under ideal standard conditions and, for most protocols, a variety of proteolytic digestion conditions has to be tested. This is due, in part, to cross-linkages between proteins, or between histones and DNA, which are difficult to remove and may impair the efficiency of PCR. Furthermore, aldehyde-based fixatives may induce single-stranded breaks that, when repaired by the DNA polymerase, result in false positivity. Fixatives containing picric acid (Bouin's solution) or mercury (Zenker's solution) seem to degrade nucleic acids and generally are not recommended (17-22). Tissues, preferablyless than 0.5 cm in thickness, are fixed at room temperature in 10 % Neutral Buffered Formalin. Most cell suspensions and cytology preparations are routinely fixed in 95 % alcohol for 1 hour either at 4^oC or at room temperature. They can be washed in PBS and kept at 4^oC for short-term storage or dehydrated and placed at –70^oC for long-term storage. Paraformaldehydealso provides excellent results, being the fixative of choice in many laboratories (21, 22). It can be used at concentrations ranging from1 % to 4 % during a period of 4 to 24 hours, preferably at 4^oC. Fixation has to be initiated as early as possible to reduce the chance of nucleic acid degradation. 3.5 Proteolytic Digestion (appendix 3) Since fixatives cross-link proteins and induce other changes that diminish the accessibility of reagents to the target, pre-treatment with proteases is necessary, particularly for paraffin-embedded, formalin-fixed tissues (17-19, 21-23). Stringent control over protease digestion is required to maintain preservation of morphology and to reduce noise to signal ratio. Therefore, a balance has to be achieved to avoid false negativity, due to poor penetration of reagents in underdigested samples, and destruction of the tissue architecture due to over-digestion by protease. It is considered that the length of proteolytic digestion correlates with the duration of fixation (8, 19). As a guideline, 10 min of digestion is necessary if tissue was fixed for 4 hours in 10 % neutral buffered formalin. On the other hand, 90 min of protease digestion may be necessary if tissue was fixed for 24 h. Bagasra et al. (15), have reported that the presence of "peppery dots", observed on the membrane of digested cells, can be used as evidence of appropriate digestion for In situ PCR. In all cases, immediately after digestion, the enzyme should be inactivated. 3.6 Additional pre-treatments (appendix 4) After proteolytic digestion, additional pretreatment steps may be necessary. When labeled nucleotides are used, reduction of the static charge of tissue sections can minimize nonspecific binding. This can be accomplished by washing the slide for five minutes in a solution of 0.1M Triethanolamine and 0.25 % acetic anhydride. If peroxidase is going to be used in the detection step, endogenous peroxidase should be quenched by any of the routinely used methods. In In situ RT-PCR it may be necessary to pre-digest the sample with RNase-free DNase, to destroy endogenous DNA.

[Frontiers in Bioscience 2, c15-29, September 1, 1997
Reprints
PubMed
CAVEAT LECTOR

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

Received 1/10/97 Accepted 7/15/97

3. GENERAL CONSIDERATIONS

3.1 Equipment

Thermocyclers are designed to reach and maintain the necessary temperatures in a reproducible manner over specified periods of time and predetermined numbers of cycles. Some of these instruments have independent "slide" blocks and "tube" blocks, connected to a common programmable controlling unit, providing the opportunity to run different protocols simultaneously and to correlate solution-PCR with In situ PCR results. The commercially available thermocyclers, use different mechanisms to prevent evaporation and increase efficiency and consistency of results. In the Omnislide Temperature Cycling System, Hybaid (Teddington, Middlesex, UK, http://www.hybaid.co.uk/), slides are placed in a sealed humid chamber. In the PTC-100-12MS Programmable Thermal Controller (MJ Research, Inc., Watertown, MA, USA, http://www.mjr.com/), the reagents are added directly to the amplification mixture to avoid evaporation. With the Gene-Amp In situ PCR System 1000 (Perkin-Elmer Corp., Norwalk, CT, USA, http://www.perkin-elmer.com/), up to 3 samples/slide can be tested by clamping slides individually using water-tight seals. Several cheaper ovens, in which In situ PCR can be performed, are also available. The BioOven from Biotherm Corp. (Fairfax, VA, USA), ensures temperature control via a sensor placed under the coverslip or glass slide that sends information to the control unit. Those that already have a conventional solution PCR thermocycler can also perform In situ PCR experiments by placing slides in a "tube" block. In these systems, however, heat transfer to the tissues is less reliable.

3.2 Technical aspects

The same precautions that apply to solution PCR procedures, should be exercised in In situ PCR experiments. Due to the extreme amplification capability of the In situ PCR, any small amount of contaminant DNA in the sample can be inadvertently amplified. During specimen handling, areas where amplified DNA (in particular previously amplified DNA) can be found should be avoided. Devices (positive displacement pipets, pipet tips containing sterile filters, tubes, etc.) have to be specifically allocated to In situ PCR experiments and frequent changes of gloves have to become routine. When working with tissue sections, appropriately coated slides (i.e., poly-L-lysine, Aminosilane) should be used to avoid the detachment of sections during the proteolytic digestion step (16-17). To avoid evaporation, a variety of methods have been attempted. The section is overlaid with the PCR mixture and a coverslip is placed over the section. The coverslip may be sealed with nail polish or rubber cement making sure not to introduce bubbles. After amplification, the seal is peeled-off with forceps and the coverslip removed in 0.1X SSC. Alternatively, specially designed cones (1, 8) are applied to the slide sealing the area of interest. The amplification solution is added to the cones making sure that it diffuses evenly over the tissue section without creating air bubbles. If the operator does not have previous experience with the procedure, it is advisable to attempt several dry runs to get acquainted with the peculiarities of the technique (slide manipulation, microscope checks, rubber cement and nail polish application and removal, transfers in and out of the thermocycler, etc.).

3.3 Types of samples (appendix 2)

In situ PCR has been successfully performed on a variety of samples: cell smears, imprints, cell blocks, cytospins, metaphase chromosomes, frozen sections and sections of tissues embedded in plastic or paraffin. Haase et al (14) performed In situ PCR on fixed cells suspended in the PCR reaction mixture within a microcentrifuge tube. In this procedure, after amplification, cells are recovered by cytocentrifugation onto glass slides (3, 4, 8, 12, 14). An advantage of this method is that cells may be lysed after amplification and the lysate analyzed by gel electrophoresis and Southern blot hybridization (14, 15). Good results can also be obtained in cytospins and cell blocks prepared following standard protocols. When tissues are used, 4-6 micron thick sections are prepared from paraffin or frozen tissue blocks. The morphological changes induced by freezing, and the increase rate of diffusion of the amplified product, make frozen sections less desirable for In situ PCR purposes. Although thicker sections may compromise evaluation of morphology and increase noise to signal ratio, it is believed that thick paraffin sections contain more starting nucleic acid material and allow a better yield of amplified signal. Deparaffinization is performed on sections cut from paraffin blocks, according to standard procedures by removing paraffin in xylene and rehydration in decreasing concentrations of ethanol.

3.4 Fixation (appendix 2)

The goal of fixation is to maintain optimal tissue morphology while preserving the integrity of the nucleic acids. It is generally agreed that aldehyde-based fixatives, such as 10% neutral buffered formalin, are superior for preservation of morphology and for minimizing the diffusion of signal (17-20). This is fortunate since the vast majority of archival material in pathology departments has been fixed in 10% neutral buffered-formalin. Nevertheless, not all routine procedures for fixation of specimens are done under ideal standard conditions and, for most protocols, a variety of proteolytic digestion conditions has to be tested. This is due, in part, to cross-linkages between proteins, or between histones and DNA, which are difficult to remove and may impair the efficiency of PCR. Furthermore, aldehyde-based fixatives may induce single-stranded breaks that, when repaired by the DNA polymerase, result in false positivity. Fixatives containing picric acid (Bouin's solution) or mercury (Zenker's solution) seem to degrade nucleic acids and generally are not recommended (17-22). Tissues, preferablyless than 0.5 cm in thickness, are fixed at room temperature in 10 % Neutral Buffered Formalin. Most cell suspensions and cytology preparations are routinely fixed in 95 % alcohol for 1 hour either at 4^oC or at room temperature. They can be washed in PBS and kept at 4^oC for short-term storage or dehydrated and placed at –70^oC for long-term storage. Paraformaldehydealso provides excellent results, being the fixative of choice in many laboratories (21, 22). It can be used at concentrations ranging from1 % to 4 % during a period of 4 to 24 hours, preferably at 4^oC. Fixation has to be initiated as early as possible to reduce the chance of nucleic acid degradation.

3.5 Proteolytic Digestion (appendix 3)

Since fixatives cross-link proteins and induce other changes that diminish the accessibility of reagents to the target, pre-treatment with proteases is necessary, particularly for paraffin-embedded, formalin-fixed tissues (17-19, 21-23). Stringent control over protease digestion is required to maintain preservation of morphology and to reduce noise to signal ratio. Therefore, a balance has to be achieved to avoid false negativity, due to poor penetration of reagents in underdigested samples, and destruction of the tissue architecture due to over-digestion by protease. It is considered that the length of proteolytic digestion correlates with the duration of fixation (8, 19). As a guideline, 10 min of digestion is necessary if tissue was fixed for 4 hours in 10 % neutral buffered formalin. On the other hand, 90 min of protease digestion may be necessary if tissue was fixed for 24 h. Bagasra et al. (15), have reported that the presence of "peppery dots", observed on the membrane of digested cells, can be used as evidence of appropriate digestion for In situ PCR. In all cases, immediately after digestion, the enzyme should be inactivated.

3.6 Additional pre-treatments (appendix 4)

After proteolytic digestion, additional pretreatment steps may be necessary. When labeled nucleotides are used, reduction of the static charge of tissue sections can minimize nonspecific binding. This can be accomplished by washing the slide for five minutes in a solution of 0.1M Triethanolamine and 0.25 % acetic anhydride. If peroxidase is going to be used in the detection step, endogenous peroxidase should be quenched by any of the routinely used methods. In In situ RT-PCR it may be necessary to pre-digest the sample with RNase-free DNase, to destroy endogenous DNA.