Microarray and Microarray Technology
Microarray is one of the most recent developments in cancer research; provides assistance in the pharmacological approach to the treatment of various diseases including oral lesions. Microarray assists in the analysis of large quantities of samples or new samples previously recorded; It even helps to test the incidence of a particular marker in tumors. Until recently, the use of microarray in dentistry was very limited, but there may be an increase in use as technology becomes available in the future. Here we discuss the various techniques and applications of microarray or DNA chip.
KEYWORDS: Cancer, human genome, microarray, microarray tissue
Writer : Osman TOKER
After the human genome sequence was completed in 2001, it led to many experiments and research; such a site was to identify the regions of DNA that control normal and disease states. Functional genomics is the study of gene function by measuring parallel expression of a genome. The most common tools used to perform these measurements include complementary DNA microarrays, oligonucleotide microarrays, or serial analysis of gene expression (SAGE). Microarray analysis can be divided into two main stages: probe production and target (cDNA) production. Specific sequences are immobilized to a surface and reacted with labeled cDNA targets. A signal from the hybridization of the labeled target with the specific immobilized probe identifies which RNAs are present in the unknown target sample.
Prevention, diagnosis and treatment in dentistry are based on an understanding of the underlying oral health and biology of the disease. Some aspects of patient care will remain untouched by the rapid developments in biological research. In the future, dentists can use cheap but remarkably sophisticated diagnostic tests to diagnose infection, oral lesions, and temporomandibular dysfunction (TMD) symptoms. Small changes in the DNA sequence that result in different properties (such as skin color, facial features or height) are also known as polymorphisms that cause or contribute to the development of many syndromes and diseases.It can be easily detected by microarray technique.
The microarray provides a basis for the genotype of thousands of different loci at a time; this is useful for coupling and connection studies to isolate chromosomal regions associated with a particular disease. This sequence can also be used to detect cancer-related chromosomal aberrations, such as allele imbalance segments, which can be identified by loss of heterozygosity.Comparative genomic hybridization techniques on genomic DNA may be expanded or deleted regions in chromosomes. In the case of oral cancer can be defined as.
What is Microarray?
- Microarray, Array Comparative Genomic Hybridization (a-CGH) is a high-tech technique for detecting small and small amounts that cannot be observed by microscopy. All chromosomes in embryos can be evaluated by microarray (a-CGH).
- DNA microarray is a series of microscopic DNA spots that are sequenced in order to create an expression profile, in other words, to a solid surface such as glass, plastic or silicon chip to simultaneously monitor the expression level of thousands of genes.
- Tens of thousands of these spots can be found in a microarray. These DNA segments (20 to 100 or more nucleotides in length) attached to the surface were identified as probes.
- Microarray technology is derived from the ‘’Southern blotting’’ in technique, which can be defined as the preparation of DNA by a known gene or fragment, which fuses with a substrate.
- Measurement of gene expression using microarrays is applicable in many areas of biology and medicine. For example, microarrays can be used to identify disease-related genes by comparing gene expression in a diseased and normal cell.
- DNA microarrays can be used to detect RNAs that can be transformed into active proteins or cannot be translated. Such assays are referred to as expression analysis or expression profiling.
- Studies on the use of microarrays for gene expression were first published in Science in 1995.
- The first eukaryotic genome completed with Microarray was the Saccharomyces cerevisiae, and the study was published in Science Journal in 1997.
- Briefly, as with all other sequencing attempts, the goal of using microarray is to answer the following question: Which genes are expressed in a particular cell of an organism at a given time and under certain conditions?
How microarray technique is produced?
- Various methods can be used in the production of microarrays:printing with needle-shaped needles on glass slides, photolithography with pre-prepared masks, photolithography with dynamic micro-devices, ink-jet printing, electrochemistry in microelectrode array …
Microarray production steps can be summarized as follows:
- RNA extraction
- Marked cDNA preparation (rev. Transc.)
- Gene array experiments are typically intended to determine the level of gene expression in different tissues or conditions or after an administration (ie, at different times). For this purpose, RNA extraction is performed based on different textures, conditions or times. These RNA samples are diluted to ensure that each sample has an even density.
- As a rule, for each mRNA molecule in the original RNA population, a single-stranded cDNA is generated that is componentistic to this molecule. The amount of cDNA increases as the density of a particular mRNA increases.
- Probes are generated by reverse transcription of mRNA to single-stranded cDNA in the presence of labeled nucleotides. Therefore, the labeled probe is actually a population of cDNA molecules representing the mRNA population.
- In order to form a single-stranded probe, RNA is introduced into a reaction containing oligo dT primers, Reverse Transcriptase and labeled nucleotides capable of forming a base pair with the polyA tip in the mRNA.
- Generally labeled nucleotides are labeled with either fluorescent markers such as Cy3 and Cy5, or digoxigenin (DIG), which can be detected by chemical luminescent detection.
- Probes are hybridized with filters containing spot-cDNAs in a two-dimensional array. The amount of hybridization in a treated clone corresponds to the amount of mRNA present for the gene of interest.
- Filter arrays are incubated with the probe and washed as in Southern or Northern blotting.
- Hybridized probe is used to detect chemical luminisens for DIG marking and direct UV fluorescence for microarrays. The line density of each spot is measured by a CCD camera and the data is a TIF is obtained as an image.
Why scientists using microarray?
- Increases or decreases in chromosome or chromosome regions can be detected in the whole genome.
- Aneuploidies can be detected.
- Changes in the amount of DNA are examined at high resolution, so it is a very reliable method.
When to apply the microarray method?
It is applied in del / dup test below 5Mb where karyotyping is insufficient.
- In the diagnosis of mental problems,
- In cases of abnormal length / shortness,
- In the diagnosis of gender-related development problems,
- It can be used in the diagnosis of developmental disorders.
- It is also possible to use aCGH method in prenatal and postnatal diagnosis:
Preimplantation genetic diagnosis (PGD) is a method used to increase treatment success and decrease pregnancy losses in couples with indications such as recurrent failures, repetitive miscarriages and severe male factors, especially when the age of the mother is advanced.
Tumor formation involves simultaneous changes in hundreds of cells and variations in genes. The microarray can be a blessing to researchers because it provides a platform for the simultaneous testing of a large set of genetic samples. In particular, it assists in the classification of single nucleotide polymorphisms (SNPs) and mutations, tumors, identification of target genes of tumor suppressors, identification of cancer biomarkers, identification of genes associated with chemoresensitivity, and drug discovery.
For example, we can compare different gene expression levels models between a group of cancer patients and a group of normal patients, and we can identify the gene associated with this particular cancer.
Gene microarrays were used for comparative genomic hybridization. In this technique, genomic DNA is labeled fluorescently and used to determine the presence of gene loss or amplification.Sequence-based comparative genomic hybridization (aCGH) was used to map genetic abnormalities in a wide variety of tumors. including breast carcinoma,bladder carcinoma,fallopian tube cancer, gastric carcinoma,melanoma,and lymphoma.
Gene expression data may identify case groups with different results when routine histopathological examination does not allow subclassification.
The transformation of a non-invasive tumor into an invasive tumor also guarantees research. Clark et al. studied the gene profile of melanoma cells that became metastatic and found a gene, expressed in RhoCmetastatic cells, compared to non-metastatic melanoma cells.
The microarray-based expression profile allows us to identify important molecular and cellular events that may be important in complex processes such as metastases as well as families of genes.
Future practical applications include diagnosis and prognostic management of patients. Clinicians will be able to use microarrays during early clinical trials to verify the mechanisms of action of drugs and to assess drug sensitivity and toxicity. They may be used to develop a novel molecular cancer taxonomy that involves clustering of cancers relative to prognostic groups based on gene expression profiles.
Areas that can be combined with microarray technologies include classifying diseases or molecular phenotyping; gene function related to gene regulatory networks or functional genomics; pharmacogenomics and developmental biology.
The increase in the number of resistant bacteria and excessive infections led to the failure of antibiotics. The virulence of bacterial strains also affects the outcome of the disease process. In the oral cavity, where anaerobic bacteria may be infective agents, these organisms such as actinomies cannot be easily developed. DNA microarray analysis usually causes bacterial genomic DNA to exceed the viability of bacteria and can be diagnosed using a small amount of DNA instead of a large number of bacteria needed for culture. In the future, an abscess sample can be sent for culture and sensitivity testing, but not for DNA microarray analysis.
Early diagnosis of oral precancerous lesions
Leukoplakia or white lesions of the oral cavity may result from numerous reversible conditions. At present, microscopic examination cannot identify small subset of these lesions progressing to oral cancer. The identification of gene expression profiles or et genomic fingerprints inin will enable clinicians to distinguish harmless white lesions from pre-cancerous lesions or early cancer. Recent studies have shown the effectiveness of microarrays in oral cancer.
In the future, samples taken from a incision biopsy or brush biopsy can be sent to a laboratory for gene expression analysis.The early diagnosis and management of oral cancer is correlated with increased survival. The identification and treatment of premalignant and early cancer oral lesions may be one of the most valuable services in their future performance.
This review gives a brief summary of the technique behind the microarray and the various steps involved. Although the technique is currently limited due to its cost factor, it can expand prospects when there is an increase in the presence of commercial products. The ability to record many old samples and analyze them for various genetic changes helps in understanding the concept of molecular biology. The microarrays are very promising for the analysis of diseases in the oral cavity. Classification of oral disease by DNA, RNA or protein profiles will greatly improve our ability to diagnose, prevent, monitor and treat patients. Today, microarrays are primarily a research tool. Microarrays promises a more biological-based, individualized and highly developed standard for oral care in the future.
How to use in ecological and evolutionary studies?
- Hihara et al. (2001) used a whole genome microarray to understand the effects of transferring from a low light condition to a high light condition in a cyanobacterial strain, Synechocystis spp., On gene expression. In this study, many new genes have been identified in addition to the genes previously shown to be associated with high light compatibility.
- The difference between injury and herbivorous insect effect in Arabidopsis was demonstrated by the transcript profile, suggesting that the feeding of the cabbage butter larvae does not produce a nonspecific stress response in plants and, perhaps, even suppress the formation of this response.(Reymond et al., 2000).
Applications of biochip and microarray systems in pharmacogenomics:
A DNA microarray system usually consists of DNA probes formed on a micro scale on a glass surface (chip) and also means (bioinformatics means) for processing (automatic robotic) samples to read reporter molecules (scanners) and analyze data. . Biochipses are formed by in situ synthesis (in DNA) or staining of DNA fragments of oligonucleotides or peptide nucleic acids (PNAs). Hybridization of samples derived from RNA or DNA in chips allows monitoring of the expression of mRNAs or the formation of polymorphisms in genomic DNA.
The basic DNA chip types are the chips for the sequence chip, expression chip and comparative genomic hybridization. Advanced technologies used in automatic microarray production are photolithography, mechanical microspot and ink jets. The bioelectronic microchips comprise a plurality of electronically active microelectrodes having specific DNA capture probes attached to the electrodes via molecular wires. Many biosensors were used with biochips.
The PNA biosensors are usually based on the immobilization of a single-stranded DNA sequence (‘probe’) to a transducer surface for hybridization with the complement (‘target’) strand to give an appropriate electrical signal. Other sensors are cell-based immuno-biosensors with designed molecular recognition, integrated biosensors based on phototransistor integrated circuits, and sensors based on surface plasmon resonance. Compared to standard gel based micro-satellite methods, microarray technologies save time and labor. Reading bioinformatics by knowledge and management is necessary because of the huge amount of data generated by various technologies using microarrays. Standardized procedures are very important for harmonious data generation, quality control and analysis.
Expression monitoring is currently the most biologically informative application of this technology. Microarray technology has important applications in pharmacogenomics: drug discovery and development, drug safety and molecular diagnostics. DNA chips will facilitate the introduction of personalized drugs, as well as the integration of diagnostic and therapeutics. Expression monitoring is currently the most biologically informative application of this technology. Microarray technology has important applications in pharmacogenomics: drug discovery and development, drug safety and molecular diagnostics. DNA chips will facilitate the introduction of personalized drugs, as well as the integration of diagnostic and therapeutics.
Expression monitoring is currently the most biologically informative application of this technology. Microarray technology has important applications in pharmacogenomics: drug discovery and development, drug safety and molecular diagnostics. DNA chips will facilitate the introduction of personalized drugs, as well as the integration of diagnostic and therapeutic
- Species microarray experiments show that it is not necessary to prepare one array for each type. Arabidopsis microarrays correspond well to the expression profile in Arabidopsis and Brassica napus seeds. You need to pay attention to a point here alone.The use of microarrays in inter-species experiments leads to inability to detect new genes that are not found in the model organism.
- Many regulatory genes (the gene responsible for events such as protein synthesis, enzyme activation, and metabolite elimination) are not present at the level of mRNA aggregation and therefore not detectable by this technology.
- In addition, the cost of microarray work is high and the profit from the solution of the ecological problem must cover this cost.
Writer: Osman TOKER
- Pollack JR, Perou CM, Alizadeh AA, Eisen MB, Pergamenschikov A, Williams CF, et al. Genome-wide analysis of DNA copy-number changes using cDNA microarrays. Nat Genet. 1999;23:41–6.
- Kononen J, Bubendorf L, Kallioniemi A, Bärlund M, Schraml P, Leighton S, et al. Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat Med. 1998;4:844–7.
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