Microarray hybridization normalization

Spike-ins are usually RNA transcripts used to calibrate measurements in a DNA microarray experiment. Each spike-in is designed to hybridize with a specific control probe on the target array. Manufacturers of commercially available microarrays typically offer companion RNA spike-ins kits . Known amounts of RNA spike-ins are mixed with the experiment sample during preparation. Subsequently the measured degree of hybridization between the spike-ins and the control probes is used to normalize the hybridization measurements of the sample RNA. 

The typical cDNA microarray study can be described in nine steps (1) establishing an appropriate experimental design (2) isolation and conversion of mRNA to labeled cDNA (3) hybridization of labeled cDNA to the microarray slide (4) image acquisition, (5) data storage, (6) normalization (7) statistical analysis (8) data mining and (9) validation of the results. Each of these steps is multifaceted and the introduction of error at any point in the process can lead to costly loss of data. The following section describes the steps followed in experimental design. 

The products of gene expression (mRNA and proteins) can be measured by techniques such as the following. Northern blots are very similar to Southern blots except that the original sample contains a mixture of mRNA molecules that are separated by electrophoresis, then hybridized to a radioactive probe. Microarrays are used to determine the differing patterns of gene expression in two different types of cells—for example, normal and cancer cells. Enzyme-linked immunosorbent assays (ELISAs) and western blots (immunoblots) are used to detect specific proteins. 

Microarray is a rapidly developing field, with the capacity to revolutionize many fields of biomolecular research. This technology promises to deliver an explosion of information on the genetic basis of normal physiological processes and disease states by providing the opportunity to simultaneously compare the relative amounts of thousands of individual sequences from complex mixtures of nucleic acids. Based on traditional nucleic acid hybridization chemistry, it offers flexibility in choice of target/probe combinations and provides quantifiable data on relative expression levels. 

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