Gene expression profiling In an mRNA or gene expression profiling experiment the expression levels of thousands of genes are simultaneously monitored to study the effects of certain treatments, diseases , and developmental stages on gene expression. For example, microarray-based gene expression profiling can be used to identify genes whose expression is changed in response to pathogens or other organisms by comparing gene expression in infected to that in uninfected cells or tissues. Chromatin immunoprecipitation on Chip DNA sequences bound to a particular protein can be isolated by immunoprecipitating that protein ChIP , these fragments can be then hybridized to a microarray such as a tiling array allowing the determination of protein binding site occupancy throughout the genome. DamID Analogously to ChIP , genomic regions bound by a protein of interest can be isolated and used to probe a microarray to determine binding site occupancy. SNP detection Identifying single nucleotide polymorphism among alleles within or between populations. Alternative splicing detection An exon junction array design uses probes specific to the expected or potential splice sites of predicted exons for a gene.
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Gene expression profiling In an mRNA or gene expression profiling experiment the expression levels of thousands of genes are simultaneously monitored to study the effects of certain treatments, diseases , and developmental stages on gene expression. For example, microarray-based gene expression profiling can be used to identify genes whose expression is changed in response to pathogens or other organisms by comparing gene expression in infected to that in uninfected cells or tissues.
Chromatin immunoprecipitation on Chip DNA sequences bound to a particular protein can be isolated by immunoprecipitating that protein ChIP , these fragments can be then hybridized to a microarray such as a tiling array allowing the determination of protein binding site occupancy throughout the genome. DamID Analogously to ChIP , genomic regions bound by a protein of interest can be isolated and used to probe a microarray to determine binding site occupancy.
SNP detection Identifying single nucleotide polymorphism among alleles within or between populations. Alternative splicing detection An exon junction array design uses probes specific to the expected or potential splice sites of predicted exons for a gene.
It is of intermediate density, or coverage, to a typical gene expression array with 1—3 probes per gene and a genomic tiling array with hundreds or thousands of probes per gene. It is used to assay the expression of alternative splice forms of a gene.
Exon arrays have a different design, employing probes designed to detect each individual exon for known or predicted genes, and can be used for detecting different splicing isoforms.
Fusion genes microarray A Fusion gene microarray can detect fusion transcripts, e. The principle behind this is building on the alternative splicing microarrays. The oligo design strategy enables combined measurements of chimeric transcript junctions with exon-wise measurements of individual fusion partners.
Tiling array Genome tiling arrays consist of overlapping probes designed to densely represent a genomic region of interest, sometimes as large as an entire human chromosome.
The purpose is to empirically detect expression of transcripts or alternatively spliced forms which may not have been previously known or predicted. Double-stranded B-DNA microarrays Right-handed double-stranded B-DNA microarrays can be used to characterize novel drugs and biologicals that can be employed to bind specific regions of immobilized, intact, double-stranded DNA.
This approach can be used to inhibit gene expression. This approach can be used to discover new drugs and biologicals that have the ability to inhibit gene expression. Fabrication[ edit ] Microarrays can be manufactured in different ways, depending on the number of probes under examination, costs, customization requirements, and the type of scientific question being asked. Arrays from commercial vendors may have as few as 10 probes or as many as 5 million or more micrometre-scale probes.
Spotted vs. The probes are synthesized prior to deposition on the array surface and are then "spotted" onto glass. A common approach utilizes an array of fine pins or needles controlled by a robotic arm that is dipped into wells containing DNA probes and then depositing each probe at designated locations on the array surface. The resulting "grid" of probes represents the nucleic acid profiles of the prepared probes and is ready to receive complementary cDNA or cRNA "targets" derived from experimental or clinical samples.
This technique is used by research scientists around the world to produce "in-house" printed microarrays from their own labs. These arrays may be easily customized for each experiment, because researchers can choose the probes and printing locations on the arrays, synthesize the probes in their own lab or collaborating facility , and spot the arrays.
They can then generate their own labeled samples for hybridization, hybridize the samples to the array, and finally scan the arrays with their own equipment. This provides a relatively low-cost microarray that may be customized for each study, and avoids the costs of purchasing often more expensive commercial arrays that may represent vast numbers of genes that are not of interest to the investigator.
Publications exist which indicate in-house spotted microarrays may not provide the same level of sensitivity compared to commercial oligonucleotide arrays,  possibly owing to the small batch sizes and reduced printing efficiencies when compared to industrial manufactures of oligo arrays. In oligonucleotide microarrays, the probes are short sequences designed to match parts of the sequence of known or predicted open reading frames. Although oligonucleotide probes are often used in "spotted" microarrays, the term "oligonucleotide array" most often refers to a specific technique of manufacturing.
Oligonucleotide arrays are produced by printing short oligonucleotide sequences designed to represent a single gene or family of gene splice-variants by synthesizing this sequence directly onto the array surface instead of depositing intact sequences. Sequences may be longer mer probes such as the Agilent design or shorter mer probes produced by Affymetrix depending on the desired purpose; longer probes are more specific to individual target genes, shorter probes may be spotted in higher density across the array and are cheaper to manufacture.
One technique used to produce oligonucleotide arrays include photolithographic synthesis Affymetrix on a silica substrate where light and light-sensitive masking agents are used to "build" a sequence one nucleotide at a time across the entire array. After many repetitions, the sequences of every probe become fully constructed. The two Cy-labeled cDNA samples are mixed and hybridized to a single microarray that is then scanned in a microarray scanner to visualize fluorescence of the two fluorophores after excitation with a laser beam of a defined wavelength.
Relative intensities of each fluorophore may then be used in ratio-based analysis to identify up-regulated and down-regulated genes. The degree of hybridization between the spike-ins and the control probes is used to normalize the hybridization measurements for the target probes.
Although absolute levels of gene expression may be determined in the two-color array in rare instances, the relative differences in expression among different spots within a sample and between samples is the preferred method of data analysis for the two-color system. Examples of providers for such microarrays includes Agilent with their Dual-Mode platform, Eppendorf with their DualChip platform for colorimetric Silverquant labeling, and TeleChem International with Arrayit.
In single-channel microarrays or one-color microarrays, the arrays provide intensity data for each probe or probe set indicating a relative level of hybridization with the labeled target. However, they do not truly indicate abundance levels of a gene but rather relative abundance when compared to other samples or conditions when processed in the same experiment. Each RNA molecule encounters protocol and batch-specific bias during amplification, labeling, and hybridization phases of the experiment making comparisons between genes for the same microarray uninformative.
The comparison of two conditions for the same gene requires two separate single-dye hybridizations. One strength of the single-dye system lies in the fact that an aberrant sample cannot affect the raw data derived from other samples, because each array chip is exposed to only one sample as opposed to a two-color system in which a single low-quality sample may drastically impinge on overall data precision even if the other sample was of high quality.
Another benefit is that data are more easily compared to arrays from different experiments as long as batch effects have been accounted for. One channel microarray may be the only choice in some situations.
qPCR, Microarrays or RNA-sequencing: When To Choose One Over the Other?
Estas secuencias son complementarias a los genes que desean ser estudiados y puede haber hasta Si se quiere comparar dos muestras sana vs. El marcaje puede ser fluorescente y debe ser diferenciable entre los dos tejidos a analizar. De manera tradicional se usan los compuestos fluorescentes Cy3 y Cy5, ya que emiten fluorescencia a distinta longitud de onda. En el caso del Cy3 es un color cercano al rojo y el Cy5 corresponde al espectro entre el naranja y el amarillo. En otras palabras, se puede conocer el transcriptoma de las muestras evaluadas en el experimento. Larssono [Public domain], from Wikimedia Commons Aplicaciones Actualmente, los microarreglos son considerados como herramientas muy poderosas en el campo de la medicina.