Homemade Microarrays

Homemade microarrays are made by printing or arraying nucleic acids (the “microarray probes”) onto pre-treated glass microscope slides. The microarray probes can either be cDNAs or oligonucleotides. To manufacture cDNA probes, cDNA clone collections are amplified by polymerase chain reaction (PCR). For oligonucleotide probes, 50-70mer oligos are ordered from a commercial vendor.

The probes are printed in a two-dimensional grid onto glass. Spots are typically 100–300 µm in size and are spaced about the same distance apart. Using this technique, arrays consisting of more than 25,000 cDNAs or oligonucleotides can be printed onto the surface of a conventional microscope slide.

The process of spotting is generally not accurate enough to allow direct comparison between different arrays. Therefore, two samples to be compared are usually bound competitively to the same array. For a gene expression experiment, RNA samples are used to generate first-strand cDNA targets. These targets are labeled with two different fluorescent dyes (for example Cy3 and Cy5) and are then pooled and hybridized to an array, resulting in competitive binding of the differentially labeled cDNAs to the arrayed sequences.