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

5. IN SITU PCR PROTOCOLS

In "direct"methods,labeled nucleotides (i.e., digoxigenin-11-dUTP, fluorescein-dUTP) are incorporated during the amplification step and then amplified product are detected by immunohistochemistry. In "indirect" methods, the amplified products are detected by In situ Hybridization (ISH) using a specific labeled probe.

5.1 In situ RT-PCR (appendix 6)

Since mRNA cannot serve as a template in PCR, a reverse transcription step is introduced to convert mRNA to cDNA (1-10). The combination of these two techniques is referred to as RT-PCR. RT-PCR may be used for the detection of mRNAs that are present at less than 10 copies/cell (1-12). The enzyme that converts RNA into c-DNA is "Reverse Transcriptase". Three Reverse Transcriptases commercially available are: the mesophilic viral reverse transcriptases from Avian Myeloblastosis Virus (AMV) and Moloney Murine Leukemia Virus (M-muLV), and rTth, a heat-stable DNA polymerase from Thermus thermophilus, that can be used to perform reverse transcription and PCR in a single step. The AMV and M-muLV RTs can reverse transcribe mRNAs up to 10 kb; rTth synthesizes cDNAs in the range of 1-2 kb. The most important factors to consider in the selection of the enzyme are: a.) RNAse H activity that degrades RNA in an RNA:cDNA hybrid, b.) temperature (M-muLV reaches maximum activity at 37^o C and AMV at 42^o C); rTth reverse transcribes mRNA at 60-70^o C and amplifies cDNA at 60-94^o C), and c.) divalent ion requirements (rTth requires manganese). Three types of primers can be used in reverse transcription reactions: a.) Oligo(dT)12-18 (it binds to the endogenous poly (A)+ tail of mRNA), b.) random hexanucleotides (these bind at any complementary sequence throughout the length of mRNA), c.) specific oligonucleotides (based on a specific mRNA sequence).

5.2 Direct In situ PCR (appendix 7)

In direct In situ PCR, the label, digoxigenin or biotin, is incorporated, into the amplified product, during the PCR step (32-36). Digoxigenin is present in Digitalis plants and therefore is not detectable in any other biological material. High affinity, unconjugated, anti-digoxigenin antibodies and anti-digoxigenin antibodies conjugated to alkaline phosphatase, peroxidase, fluorescein or rhodamine, are readily available (37). Biotin is a member of the vitamin B complex and can be detected with an anti-biotin antibodies (37). The final result is the enzymatic localization of a chromogen at the site of DNA amplification. Due to its simplicity, direct In situ PCR represents a logical procedure of choice. However, in the experience of many investigators (5, 8, 24, 35), direct detection procedures tend to yield false-positive results (35-36) that may be due to either nonspecific incorporation of labeled nucleotides into fragmented endogenous DNA undergoing "repair" by the DNA polymerase (DNA-repair artifact), or nonspecific priming by cDNA or DNA fragments (endogenous priming) (35-36). The false positive signal is typically present in the nucleus, particularly in apoptotic and senescent cells where DNA fragmentation occurs (26, 29-31). This endogenous DNA amplification is difficult to avoid, even by DNAse pretreatment (5, 35, 36). Several alternatives to reduce the artifact have been attempted with only partial success. These include the use of exonuclease-free DNA polymerase (5, 36-38), repair of DNA nicks by treatment with T4 ligase (39-44), or initial thermal cycling using unlabeled nucleotides (44, 48).

5.3 Indirect in situ PCR (appendix 8-9)

The indirect method, although more cumbersome, provides an extra level of specificity since the probe is designed to be complementary to an internal sequence within the amplified product (39, 48, 49). Double-stranded DNA, single-stranded DNA, oligonucleotides 20-30 bases long, and single-stranded RNA have all been successfully used as probes for the detection of amplified products (30). Hybridization of probes to the amplified product follows the general rules described for In situ Hybridization (ISH) procedures (50). The kinetics of the reaction are influenced by: a.) accessibility of the target (in In situ PCR methods, the target is readily accessible because hybridization takes place after DNA amplification), b.) concentration of the probe, and c.) stringency of hybridization. A series of parameters influence the stability of the hybrids: a.) T_m (formamide decreases the T_m allowing use of lower temperatures to achieve a higher stringency), b.) base composition (the greater the G+C content the higher the T_m), c.) sequence identity between the probe and the target, and d.) composition of hybridization/washing solution (higher concentrations of monovalent cations increase the stability of hybrids). The final sensitivity depends on the method of probe labelling and detection. Labelling of the probe can be done with radioactive labels ([³H], [³⁵S], [³²P], [³³P]. Factors to be considered are cost, instability, and biohazard potential of the radioisotope used. Alternatively, the probe can be labeled with non-radioactive haptens, such as biotin, digoxigenin or FITC. The efficiency of the non-radioactive detection methods has been recently improved using immunogold silver detection methods (38, 51).