As anyone who’s done some heavy hybridizing lately knows, the cost of an Affymetrix experiment involves much more than chip prices: assembling the various reagents can leave experimenters with a bigger bill than they expected.
Enter Regon Molecular Systems, a company being launched by Chris Dyanov, director of the University of Chicago’s gene arrays and expression lab. Dyanov has cooked up a mix of home-brew reagents for Affymetrix experiments that he said saved him 50 percent over the cost of an Enzo BioArray reagent kit, and also time, while increasing their enzymatic reaction yields and making the arrays more sensitive to low-level expression.
”There’s no better kit on the market than the Enzo in the amount of product synthesized, and in the label incorporation, but our version yields 5 to 10 [times] higher product,” said Dyanov. Additionally, with the in vitro transcription (IVT) step, ”we went from $35 per reaction to $5 per reaction.”
This process of developing these reagents involved ”hundreds of gene chip array hybridizations on different species,” he said. Dyanov and his colleagues tested the IVT reaction components with T7 polymerase, and found the USB polymerase fragmented the cRNA. They found a custom vendor that ”significantly increased our yield,” he said.
They also found their own universal housekeeping genes that maintained no variation in their expression levels for the kit. In one study, Dyanov sought to improve labeling efficiency of sample by incorporating three and then four NTPs for biotinylating, and also trying different NTPs than the ones the Enzo kit uses. ”From theoretical considerations we expected four NTPs would perform better,” he said. But he found that when more NTPs were added to the mix, the proportions of the various ingredients had to be changed. While he did find that the four-NTP labeling reactions were more sensitive, Enzo has stopped selling his lab the NTPs after finding out what they were doing, and so the company will not be able to commercialize this recipe.
Dyanov also has conducted comparison studies of his reagents and the Enzo kit using Affymetrix U74A chips. Although these chips contained improper sequences, they worked as test arrays because he was not concerned about the underlying meaning of the signal – just the efficiency and consistency of hybridization. In the studies, he performed reverse transcription, labeling, and in vitro transcription (IVT) for one part of a sample with Enzo reagents and used his kit to do the same for the other half of the sample. He found they had the same performance.
Regon will be selling general kits when it launches formally in October or November, said Dyanov. ”I am probably going to separate [the reagents] into two kits. I will be selling everything from the reverse transcription and everything else for the Affymetrix system as a [unit] except the IVT reaction. The IVT reaction I may sell separately.”
Dyanov has not yet determined the price of this kit, but said he will sell it online, and that it is going to be ”cheaper than anything else” on the Affymetrix reagent market.