Author: Rodney E. Shackelford, DO, Ph.D. (see Reviewers page)
Revised: 3 July 2010, last major update July 2010
Copyright: (c) 2008-2010, PathologyOutlines.com, Inc.
● Although there are many different materials and variations commonly used in microarray, the basis of the technique involves the binding of nucleic acid “probes” or “capture molecules” to a solid support
● These nucleic acids are often chemically synthesized oligonucleotides or cDNAs derived from mRNA, which are labeled with an appropriate fluorescent or luminescent-labeled probe, such as Cy3, Cy5 or dye-doped silica nanoparticles
● cDNA probes are usually 25-65 base pairs and often derived from the 3’ end of mRNAs, as many of these sequences were captured from transcripts using polyT primers
● Oligonucleotide probes are usually larger, often 1,000 nucleotides
Measuring gene expression
● Measuring gene expression via cDNA microarray is referred to as “expression analysis” or “expression profiling”
● The labeled nucleic acid is bound via nucleic acid derivatives attached to the solid support by S-carboxymethyl-L-cystein, polyacrylamide, 3-aminopropyltrimethoxysilane, 3’ glycidoxy propyltrimethoxysilane, phenylisothiocyanate, 1-ethyl=3-(3-dimethylaminopropyl)-carbodiimide hydrochloride, or other compounds
● The linkages themselves must be freely soluble in the hybridization and washing buffers, chemically stable, and long enough to prevent inhibition of probe-target nucleic acid hybridization by steric hindrance
● Microarray can use up to four different labels; gene expression profiles use one or two probes
● When one probe in employed, the sample (treated) and control (untreated) are hybridized to separate probe sets and compared; when the sample and control are differently labeled, a single slide is used
● The solid support is commonly a glass slide, silicon biochip or nylon membrane, often called a gene chip
● The bound nucleic acid probes may be synthesized oliogonucleotides, cDNAs or more rarely RNA
● Typically the amount of each bound probe is a few picomoles
● Each probe hybridizes with a different fluorescently labeled target nucleic acid species, such as an mRNA derived cDNA
● The hybridization occurs via complementary Watson-Crick hydrogen bond formation between the A:T and G:C pairs
● Binding specificity is increased with increased bp length of the probe-target hybridization sequence, a high probe-target G:C content and bp complementarity, and if possible, the same/similar probe-target Tms (sometimes be difficult with shorter cDNA-derived probes, usually not a problem with longer oligonucleotide probes which usually have similar Tms)
● In some cases mixing is employed
● Mixing can be done by magnetic bar stirring, air driven bladders or centrifugal/shear mixing
● Mixing often requires some sample dilution, however it reduces hybridization time, increases signal, lowers background, and provides homogeneous hybridization conditions
● The ratio of labeled target to probe is very high in microarray, so high that the amount of target that actually hybridizes with the probe is less than 1% of the total target molecules.
● Thus probe-target hybridization does not result in significant target dilution which could alter assay results
Maintaining high sensitivity and specificity
● Once binding is completed, the chips are extensively washed under stringent conditions, resulting in only exact or near exact Watson-Crick base pairing
● High specificity is further achieved by having the hybridization step occur at a relatively high temperature, with a low buffer salt concentration
● High stringency prevents the binding of noncomplementary strands, hairpin formation (probe or target self-hybridization) and the disassociation of strands with very high complementary
● High sensitivity is achieved by the target nucleic acid sequences being highly concentrated
● Hybridization of a labeled target sequence reveals target binding only
● The size, sequence and composition of the target are unknown.
● Labeled target sequences are measured via scanning, with images analysis performed by specialized software
● Fluorescent intensity is measured and quantified, with the darkest areas (pixels) equal to no signal, the brightest or “whitest” areas recorded as maximum intensity, and intermediate “grey” areas given corresponding signal intensity
● Defining the areas to be quantified is difficult and can be done by the user circling the spot, or by the software program
● Once a spot is defined, the total signal intensity is summed and divided by the number of pixels within the spot to give the total signal intensity
● The average background of the slide is often subtracted from the value of each spot to give initial data
● The final data is derived from various statistical analytic methods
● Microarray results show only the relative gene expression levels, not the absolute amount of a gene expression
● It is also a limited “snapshot” of gene expression patterns and multiple microarray studies or other molecular methods are required to examine changes in gene expression patterns over time.
● Additionally, it is often prudent to verify microarray results with other techniques (PCR, Northern blotting or RNase protection assays are often used)
End of Molecular Pathology > Microarray > Basic procedures
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