In microarraying as in diplomacy, protocol is everything.
Or at least it''''s very important, according to John Quackenbush of the Institute for Genomic Research''''s eukaryotic genomics department. Quackenbush and his colleagues at TIGR, who are conducting large-scale experiments with human colon cancer, mouse, and Arabidopsis microarrays, have just published a new updated version of their arraying protocol. The protocol, which includes template preparation steps, PCR amplification and purification, printing in dimethyl sulfoxide (DMSO), amino-allyl labeling, and hybridization, can be found on the TIGR website, at http://www.tigr.org/tdb/microarray/ protocols.shtml.
In this protocol, TIGR researchers use a secondary coupling technique to label their RNA, in which the reverse transcription (RT) step incorporates nucleotides coupled to an active linker arm ó but without the bulky fluorescent dyes. The dyes are covalently coupled to the labeled cDNA in a separate reaction. This technique allows for more efficient labeling, offers the advantage of allowing researchers to make labeled cDNA and store it indefinitely, and also minimizes differences that have been frequently reported between Cy3 and Cy5 label detection, said Quackenbush.
"The reverse transcriptase commonly used to make nucleotides doesn''''t have to deal with this big bulky dye, so we get better performance," Quackenbush said. "This method has at least halved our cost of labeling the reaction, and allowed us to use smaller amounts of RNA."
The Cy3-Cy5 difference, he said, is partially caused by the fact that reverse transcriptase differentially incorporates the dyes. So when the labels are added later, the difference is largely eliminated, according to Quackenbush. However, "the Cy5 is still a little finickier than Cy3," he added.
Another feature of the TIGR protocol is its simple pre-hybridization step. This step involves preparing a pre-hybridization buffer of 1 percent bovine serum albumin (BSA), with 5x SSC and 0.1 percent sodium dodecyl sulfate, then dipping the slide in the buffer. "It works phenomenally well, it is inexpensive, and very easy," said Quackenbush. "People have told me this is the most important thing we''''ve done for microarrays."
The protocol also endeavors to improve the microarray signal by spotting down a mixture of 50 percent DMSO and 50 percent PCR product rather than using sodium citrate (SSC) with the PCR; and to use amine silane slides rather than polylysine slides. "The glass is crucial!" states a PowerPoint slide in TIGR''''s microarray protocol presentation, which is also available at the website.
Currently, TIGR''''s substrate of choice is the TeleChem Super Amine Slide, said Quackenbush. "They have a very good signal-to-noise ratio and almost no background."
With these and further protocol improvements, Quackenbush believes microarray experiments can become effective for the smaller and smaller samples that current experiments with disease tissues allow.
"I think we can go a long way towards reproducibly labeling and hybridizing with sub-microgram quantities of RNA," said Quackenbush. "It''''s a question of how much time you can spend optimizing things and how much time you want to spend generating data."
Meanwhile, the TIGR group is working on adding to its offerings of analysis software for microarrays, in addition to the currently available Spotfinder and Multiexperiment Viewer (MeV), and expects to announce new tools soon.