A microarray is a collective term for modern molecular biology assay systems that allow the parallel analysis of several thousand items of evidence in a small amount of biological sample material. There are several forms of microarrays, sometimes referred to as “gene chips” or ” biochips “, because, like a computer chip, they can contain much information in a small space.
In the tissue microarrays (TMA) punched tissue cylinders of different origin are assembled on a paraffin block. Depending on the size of the punch, usually between 0.6 mm and 2 mm in diameter, can accommodate between 50 and 400 samples on a 1.5 × 3 cm large area while z. B. by immunohistology to be examined. With this method, for example, on a slide with a single application of an antibody numerous samples (eg tumors of different origin) can be examined. The advantage here is the low material consumption with a large number of received data sets. The disadvantage may be that the punched out tissue section is not representative of the entire tissue. However, this disadvantage usually arises only in complex tissues (eg liver). In the usual application with tumor material this problem is to be neglected, since it does not depend in the application of the TMA on the individual result, but the results of the investigation collective. In addition to the application in immunohistology are also analyzes by means of in situ hybridization possible (FISH, CISH).
The microarray can be manufactured using different technologies, such as the printing of microgrooves, with a particular micro-spiked pointed on a glass plate where covalently the probe (probe) of genetic material obtained by cloning using the PCR technique ( photolithography ) will be attached.
Every single clone is placed in the exact position on the slide by a robot. It is clear that this technique requires very sophisticated robotic equipment. The nucleus of the apparatus consists of a “writing group” that picks up one or more samples of cDNA through the use of nibs and transfers them on microscope slides, the movement is obviously controlled by a computer. During the deposition the robot control system automatically registers all the information necessary for the characterization and complete identification of each point of the matrix. Once the probe is on the slide, processing is performed, the passage in which the probe is covalently linked to the support through a reaction triggered by irradiation with ultraviolet light or by incubating the slide at 80 C for 2 hours. Finally, the cDNA is made single chain through a thermal or chemical denaturation. With this technique, however, it was possible to create only low-density microarray (that is, with few probes per square mm). DNA microarray can be used to detect RNA that may or may not be translated into proteins. Scientists call this analysis “expression analysis” or expression profile. With microarray technology, you can have tens of thousands of results in no time. For this reason, this technology has allowed considerable acceleration in different fields of biochemical and biotechnological investigation.
The protein -Microarray as well as a DNA microarray includes a plurality of test fields in confined spaces. However, in the protein microarray in each test field – also called Spot – small amounts of protein are fixed on the carrier material. The mocking process called for because of the small test areas with a small distance high precision and is therefore performed by special equipment.
Either a purified protein, for example, an antibody, or a protein mix of the tested sample can now be applied to the array. Those spots in which no interaction takes place remain empty after a washing step has been carried out. The detection method then allows the distinction between spots with and without protein-protein interaction. Quantitative detection methods are also possible in which the amount of adherent protein can be determined.
Types of Protein Microarrays
One can differentiate the different protein Microarrays kinds after the kind of the interaction ( antigen – antibody, enzyme – substrate, receptor protein or general protein-protein interaction). It can also be differentiated whether proteins of the sample are fixed to the array and then tested with a variety of specific, known test proteins – or whether the test proteins are fixed in the test areas and then the reaction with the sample proteins takes place.
- The reverse-phase protein microarray method (also called lysate microarray) is used to detect antigens in cell lysates of various tissues or in protein fractions obtained by isoelectric focusing. The cell lysate or the protein fraction is spotted on the carrier material of the microarray, after which the antibody is applied. In each test field with antibody-antigen interaction, the antibody remains attached. Fields with antibodies can then be used as in western blotting detected. This is usually done via a labeled second antibody that binds the antigen-specific first antibody. This second antibody is then coupled to a fluorescent or near infrared dye and detected with a corresponding scanner, or is coupled to an enzyme, horseradish peroxidase, which permits a light emitting reaction or color reaction (use of chromogens) for detection. Lysate microarrays allow the detection and quantitation of one antigen in many different lysates simultaneously. This method is limited only by the limited amount of specific antibodies required for the precise detection of a specific antigen.
- Antibody Microarrays: The antibodies are fixed (spotted) and then the sample (eg, complex cell lysates) is applied to the array. The antigen binds to the respective immobilized antibody (so-called catch antibody). These trapped antigens must now be detected with a second specific antibody (detection antibody), which is then either self-labeled or detected with a labeled secondary antibody. This complex is then detected by the label and quantified.
- Antigen Microarrays: A different antigen is fixed on each test area of the array. If the serum of a blood sample contains the corresponding specific antibody, it will adhere to the test area. Thus, the response to a variety of bacterial antigens or allergens can be tested simultaneously. The first antibody is bound by a labeled second antibody in a further incubation step and can be detected.
- In protein domain microarrays, fusion proteins are fixed on the array to detect protein-protein interactions. The fusion protein allows reliable fixation on the array with the first part without disturbing the interactivity of the other part of the protein. The applied protein will only adhere to those test areas where interaction occurs.
- A peptide microarray contains short peptide sequences, which can either be synthesized in situ, depending on the method or applied directly to the surface using a laser printer and solid-phase synthesis , . This method has several advantages, including a. lower synthesis costs and a larger number of peptides that can be printed in parallel. Peptide microarrays are u. a. used for profiling of enzymes, for the study of antibody epitopes ( epitope mapping), or to elucidate the amino acids necessary for protein binding. In practice, peptide microarrays u. a. for the monitoring of therapeutic interventions, stratification of patients, for the profiling of immune responses of individual patients as the disease progresses, or for the development of diagnostic and therapeutic agents and vaccines.
A potential advantage compared to DNA microarrays is the faster on-site analysis of samples, since one can do without the often necessary amplification of genetic material as well as the hybridization. In addition, protein microarrays allow high-throughput analysis of protein levels. Recent research suggests that mRNA and protein levels do not always correlate with each other. Thus, cDNA microarray results are not necessarily indicative of protein expression.