The Basics: What is a Gene Array?
|Gene arrays are solid supports upon which a collection of gene-specific nucleic acids have been placed at defined locations, either by spotting or direct synthesis. In array analysis, a nucleic acid-containing sample is labeled and then allowed to hybridize with the gene-specific targets on the array. (In the literature, the term "target" can refer to either the nucleic acids attached to the array or the labeled nucleic acid of the sample. In this article, the nucleic acids attached to arrays are called "targets," whereas the labeled nucleic acids comprising the sample are called "probes.") Based on the amount of probe hybridized to each target spot, information is gained about the specific nucleic acid composition of the sample. The major advantage of gene arrays is that they can provide information on thousands of targets in a single experiment. |
Currently the solid supports upon which nucleic acids are arrayed are either glass slides or nylon membranes. Typically, fluorescently labeled probes are used with glass arrays, while radiolabeled probes are used with membranes. Depending on the type of array, the arrayed nucleic acids may be composed of oligonucleotides, PCR products or cDNA vectors or purified inserts. The sequences may represent entire genomes and may include both known and unknown sequences or may be collections of sequences such as apoptosis-related genes or cytokines. Many pre-made and custom arrays are available from commercial manufacturers although many labs prepare their own arrays with the help of robotic arrayers. The methods of probe labeling, hybridization, and detection depend on the solid support to which the sequences are bound.
Many terms exist for naming gene arrays, including biochip, DNA chip, GeneChip® (a registered trademark of Affymetrix, Inc.), DNA array, microarray and macroarray. Generally when biochip, DNA chip or GeneChip is used, it refers to arrays on glass supports. Microarray and macroarray may be used to differentiate between spot size or the number of spots on the support. Gene arrays may be used for sequence identification (e.g. mutation analysis) or differential expression analysis of two or more RNA samples. This discussion will focus on the use of arrays for expression analysis.
Gene Arrays for Expression Analysis
- Isolating RNA from the samples to be compared
- Converting the RNA samples to labeled cDNA via reverse transcription; this step may be combined with aRNA amplification
- Hybridizing the labeled cDNA to identical membrane or glass slide arrays
- Removing the unhybridized cDNA
- Detecting and quantitating the hybridized cDNA, and
- Comparing the quantitative data from the various samples
Some array manufacturers offer custom analysis services and may perform the probe labeling and hybridization reactions as a service.
Nylon Membrane Arrays
Glass Slide Arrays
For membrane array analysis, a file of the data is generated by phosphoimaging and that file is then analyzed using software. The software will correlate spots to genes and can compare spot intensities for differential expression studies.
Glass array data is treated in much the same way, but the image's fluorescence is scanned and the software allows detection of each samples' fluorescence individually or simultaneously for analysis. Most software packages can analyze several arrays simultaneously.