In-situ PCR, or in-situ polymerase chain reaction, is a technique used in scientific research. Each new technology developed in science brings forth a series of new research findings, thereby driving the advancement of various disciplines.
The development of In-situ PCR
- In the 1950s, the introduction of electron microscopy into morphological observation brought about in-depth research from the cellular level to the subcellular level.
- In the 1960s and 1970s, the widespread application of immunohistochemistry and immunocytochemistry techniques pushed observation from subcellular structures to the level of protein molecules, allowing the localization of numerous active substances within cells or at the subcellular level, profoundly impacting the development of medical biology.
- In the 1970s, the extensive application of molecular biology techniques in morphology, along with the emergence of in-situ hybridization technology, enabled the localization of specific DNA or RNA sequences within tissue cells, raising the level of observation and localization from proteins to the genetic level, namely nucleic acid molecules. This deepened human understanding of many biological phenomena at the genetic level.
- In the 1980s, PCR (polymerase chain reaction), a robustly vital technique in the field of molecular biology, emerged. It was quickly introduced into the field of morphological observation, enabling the localization and observation of specific DNA or RNA with low copy numbers or single copies within cells. The advent of this technique is bound to bring forth more research findings, propelling morphological studies forward by a significant stride.
The principles of In-situ PCR
- Basic Principle: In-situ PCR combines the high-efficiency amplification of PCR with the cellular localization of in-situ hybridization to detect specific DNA or RNA sequences within tissue cells.
- PCR Technique: Utilizes DNA polymerase to amplify specific target sequences through denaturation, annealing, and extension cycles, resulting in highly sensitive and specific amplification.
- Limitations of Traditional PCR: Traditional PCR is conducted in a liquid phase, requiring cell disruption and nucleic acid extraction before amplification, making it challenging to correlate PCR results with cellular morphology and identify cell types containing specific target sequences.
- Advantages of In-situ PCR: Combines the strengths of PCR and in-situ hybridization techniques while overcoming their respective limitations.
- Procedure: Tissue samples are chemically fixed to maintain cellular morphology. The permeability of cell and nuclear membranes allows PCR components to enter cells or nuclei, where they amplify RNA or DNA fixed in situ. Amplified products, typically large molecules or interwoven structures, are retained in situ and easily detected by in-situ hybridization.
- Benefits: Enables the exponential amplification of specific DNA or RNA sequences within cells, facilitating their detection via in-situ hybridization.
Basic Types of In-situ PCR
In-situ PCR techniques can be categorized into two main types: direct method and indirect method, based on whether the triphosphate nucleotides or primers used in the amplification reaction are labeled. Additionally, there is the reverse transcription in-situ PCR technique.
Direct Method In-situ PCR Technique:
- In the direct method, the amplification products are directly labeled with marker molecules, such as labeled triphosphate adenosine or primer fragments. During PCR amplification of the specimen, the labeled molecules are incorporated into the amplified products, enabling the visualization of specific DNA or RNA in the specimen (in-situ).
- Common markers include radioactive isotopes like 35S, biotin, and digoxigenin. These markers’ locations can be detected using methods such as autoradiography for radioactive isotopes or affinity tissue chemistry and immunohistochemistry for biotin and digoxigenin.
- Advantages of the direct method include simplicity, shorter processing time, and ease of operation. However, it has disadvantages such as lower specificity, higher likelihood of false positives, and lower amplification efficiency, especially on paraffin sections where DNA damage during sectioning can lead to false positives.
- Indirect method In-situ PCR Technique:
Indirect Method In-situ PCR Technique:
- In the indirect method, specific DNA or RNA amplification is first performed within cells, followed by in-situ hybridization using labeled probes, significantly improving specificity. It is currently the most widely used in-situ PCR technique.
- Unlike the direct method, the reaction system is similar to conventional PCR, with no labeled primers or triphosphate adenosine used. The purpose is to amplify first, then detect the specific DNA products amplified within cells using in-situ hybridization technology. It effectively combines PCR and in-situ hybridization techniques, also known as PCR in-situ hybridization (PISH).
- The advantages of the indirect method include higher specificity and amplification efficiency, but it involves more complex operational steps than the direct method.
In-situ Reverse Transcription PCR Technique:
- In-situ reverse transcription PCR (In Situ RT-PCR) is a new technique that applies liquid-phase RT-PCR technology to tissue cell samples. Unlike liquid-phase RT-PCR, tissue samples are treated with DNA enzymes before in-situ reverse transcription PCR to destroy tissue DNA, ensuring that the amplified template is cDNA synthesized from mRNA, not the original DNA in cells. Other basic steps are similar to liquid-phase RT-PCR.
How to Run In-situ PCR Test?
The basic steps of in-situ PCR technology include sample preparation, in-situ amplification (PCR), and in-situ detection, as outlined below (with a focus on paraffin sections):
Sample Preparation:
- In-situ PCR techniques can be applied to cell suspensions, cell smears, frozen sections, and paraffin sections.
- Compared to other methods, in-situ PCR yields the best results with suspended intact cells, while paraffin sections yield the poorest results.
- Some reasons for poor performance include:
- Poor thermal conductivity and uneven heat convection when conducting PCR on slides.
- Taq DNA enzyme adsorption to glass slides.
- After sample processing, cells lack intact cytoplasm or nuclear membranes, leading to diffusion of amplification products and difficulty retaining them in situ.
- Most pathology specimens are fixed with formalin and preserved in paraffin.
- Solving technical issues related to in-situ PCR on paraffin sections would be highly significant.
Tissue Cell Fixation: Tissue cells are usually fixed with 10% buffered formalin or 4% paraformaldehyde for better results in in-situ PCR. The fixation time should not be excessively long, typically 4-6 hours at 4°C.
Slice Thickness: Thicker slices generally yield better results in in-situ PCR because they contain more target DNA and membrane structures, preventing diffusion of amplification products. However, thicker slices may lead to more cell overlap and decreased resolution in morphological observation.
Slide Treatment: To prevent detachment of paraffin sections during PCR and in-situ hybridization, slides should be treated to prevent detachment. Common methods include coating with poly-L-lysine or silanization treatment, which generally prevent tissue detachment.
Protease Digestion:
In-situ Amplification (PCR): In-situ amplification involves conducting PCR reactions on tissue cell specimens, with the basic principle being the same as that of liquid-phase PCR. Primers used in PCR are generally 15-30bp, and the amplified fragments are around 100-1000bp.
Reaction System:
- The reaction system for in-situ PCR is similar to that of conventional liquid-phase PCR.
- Higher concentrations of primers, TaqDNA polymerase, and Mg2+ are suggested for better amplification results on fixed tissue slices compared to liquid-phase PCR systems.
- Bovine serum albumin (BSA) should be added to the reaction system to prevent Taq DNA polymerase from binding to glass slides and reduce amplification efficiency.
Thermal Cycling:
- Thermal cycling for in-situ PCR can be done in a specialized thermal cycler or a regular PCR thermal cycler.
- Each step in the thermal cycling of in-situ PCR may be slightly longer than in conventional PCR to ensure adequate amplification.
- A hot start method can be employed to minimize the loss of the reaction system during thermal cycling.
Washing:
- After in-situ amplification, samples should undergo washing to remove amplification products that have diffused outside cells.
- Inadequate washing may lead to visualization of amplification products during detection, resulting in excessively dark backgrounds or false-positive results.
- Excessive washing can also result in the removal of amplified products inside cells, weakening or losing positive signals.
Post-fixation: Some authors have reported using 4% paraformaldehyde for 2 hours or 2% glutaraldehyde for 5 minutes for post-fixation after amplification to ensure that the amplified products are well retained in cells during detection, thereby improving sensitivity and specificity of detection.
In-situ Detection: The method for detecting amplification products of in-situ PCR depends on the design of the in-situ PCR protocol. Direct methods directly detect the amplified products based on the nature of the labeled molecules, while indirect methods require detection through in-situ hybridization.
The application of In-situ PCR
Detection of Exogenous Genes
- Detection of Viral Genes:
- In cells infected with viruses, detection can be challenging. However, in-situ PCR offers hope in addressing this difficulty.
- In-situ PCR has been successfully applied to observe the role of various viruses such as HIV, HPV, HSV, HBV, HCV, etc., in conditions like AIDS, reproductive system tumors, hepatitis, and liver cancer, enabling the timely identification of infected individuals.
- Detection of Bacterial Genes:
- A prominent application is in the detection of Mycobacterium tuberculosis. When tuberculosis lesions are atypical, it’s challenging to identify the bacteria under the microscope using special staining methods. In-situ PCR can assist in making a definitive diagnosis, even when the presence of Mycobacterium tuberculosis is minimal.
- Detection of Imported Genes:
- In the study of transgenic animals, confirmation of gene importation, as well as in patients undergoing gene therapy, can be verified using in-situ PCR. Thus, in-situ PCR has become an important detection method.
Detection of Endogenous Genes
Detection of Abnormal Genes:
- Mutation and Rearrangement Detection:
- In-situ PCR is capable of detecting mutations and rearrangements in genes within the body.
- Mutations in proto-oncogenes and tumor suppressor genes, as well as rearrangements of immunoglobulin heavy chain genes, are examples of genetic alterations detected using in-situ PCR.
- Applications in Cancer Research and Diagnosis:
- These mutations and rearrangements play crucial roles in cancer development and progression.
- In-situ PCR offers broad applications in cancer research and diagnosis by enabling the precise detection of these genetic alterations.
- It facilitates the understanding of molecular mechanisms underlying cancer and aids in the development of targeted therapies.
Detection of Constitutive Genes:
- Challenges of Low-Level Gene Expression:
- Constitutive genes expressed at low levels pose challenges for detection methods.
- In-situ hybridization techniques are ineffective due to the limited gene copy numbers in body cells.
- Limitations of Liquid-Phase PCR:
- Liquid-phase PCR can amplify and detect low-level genes but lacks cell type specificity.
- It cannot determine which cell type contains the target gene accurately.
- Advantages of In-situ PCR:
- In-situ PCR overcomes these limitations.
- It enables the detection of various human genes, even those expressed at low levels.
- In-situ PCR contributes to the completion of the human gene map by providing accurate gene localization within specific cell types.