Dot Blot Arrays

The probes for "dot-blot" slides are created from cDNA clone libraries using the polymerase chain reaction to amplify part of the cDNA clone insert of each bacteriophage in the library. The "arrayer," a robot, arrays 0.25-1 nL of probe cDNA solution in the same place on the 20 or more chemically treated slides under production. As many as 40,000 different probes can be arrayed per standard size slide. After the probe solutions have dried, the probes are covalently fixed to the slide using ultraviolet light. Excess probe is then washed off using 0.1% sodium dodecyl sulfate) Depending on the number of probes held, a batch of slides takes anywhere from 1 to 8 h to make. Maintaining the clone libraries required for dot-blot slides makes slide manufacture too expensive for all but the largest noncommercial organizations, unless the number of probes arrayed is compromised, and has sensibly lead to the establishment of core facilities capable of providing arrays to a network of labs. Nonetheless, the resultant slides are cheaper to produce than oligonucleotide arrays. A concern with cDNA arrays is the clone error rate, that is, when a clone transpires not to contain the plasmid sequence it is meant to. In one quality-control study, of the 1189 clones sequenced, only 62% were uncontami-nated and contained cDNA inserts that had significant sequence identity to published data for the ordered clones (9).

Because the volume of each probe spot deposited can vary, apparent expression changes could be produced by spot differences alone. To compensate for this artifact, two target samples are used: a test sample and a control or reference RNA, usually created from pooled cell lines in sufficient quantity to be used with all the test samples in a particular experiment. The test sample RNA

is converted into labeled cDNA target using one fluorophore whereas the reference RNA is converted into labeled cDNA target using a different fluorophore. Equal amounts of test and control target are added to each array, allowing the test and reference cDNA fragments to compete to hybridize with the probes. If a test cDNA is in excess the resultant signal will come from its fluorophore, whereas if the control cDNA dominates, the fluorescence detected will come from the control cDNA. Thus, the problem of dot variability is overcome by using a competitive hybridization method. For a comprehensive guide to the manufacture of dot-blot arrays see the protocol pages of http:// www.microarrays.org/index.html. cDNA arrays can be purchased from Agilent (http://www.chem.agilent.com/) as well as other suppliers.

1.3.1. Oligonucleotide Ink Je/Piezo Arrays

A development from dot-blot arrays, oligonucleotide ink jet/piezo arrays use no-contact, less-wasteful piezo electric pulse or ink-jet technology to propel a tiny volume of synthesized oligonucleotide solution on to the platforms. Oligomers of 60 base-pair lengths were found to offer optimal sensitivity and specificity (10). Sensitivity down to one transcript per cell can be achieved. Standard and custom-made 25- or 60-mer oligonucleotide arrays are available from Agilent (http://www.chem.agilent.com/) and Amersham Biosciences (http://www4.amershambiosciences.com/). Whether the additional cost (compared with standard cDNA arrays) is truly outweighed by more informative data production remains to be decided.

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