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ERCC Gears up for Final Testing; Reference RNA Controls Could be Available Next Year

The External RNA Controls Consortium is entering a final arc of testing that could make RNA controls generally available by the second half of next year, according to Marc Salit, ERCC chairman.
Salit, a research chemist at the National Institute of Standards and Technology, replaced Affymetrix’s Janet Warrington as chair of the ERCC in June. He told BioArray News last week that the consortium narrowed a set of 144 control RNAs down to 106 controls in initial testing, and that the ERCC plans subsequent phases of testing over the next six months to produce a final set of 96 RNA reference controls that could become available through NIST by mid-2008.
“We have demonstrated that 106 of the 144 we tested in the first round of tests performed sufficiently well to move into this next round,” Salit said. According to Salit, the next phase of testing will involve a Latin square design to establish dose response behavior to the control transcripts. The ultimate goal is to have a plasmid DNA library available to users like core labs or commercial vendors that will use the library to manufacture RNA controls.
Salit said that the ERCC expects to have results from the second phase in late summer. “At that point what we will do is to complete … the selection of the 96 [controls] from the 106, and we’ll have a couple of series of tests while the manufacturers are refining probe content and we are refining [the] reference material,” he said.
Since the ERCC’s inception in 2003, participants from industry, the government, and academia have sought consensus on spike-in RNA controls that will enable scientists to gauge the performance of the microarray assays they are running. The consortium has decided to use synthetic mammalian RNA sold by NIST as reference material, and platform manufacturers have agreed to add probes to their arrays that will detect the control RNA without interfering with existing content.
The road to achieving the RNA controls has taken over four years. During that time, the target date for when the reference material would become available has shifted from mid-2006, and later to the end of 2007, and now to mid-2008. In 2005, the Clinical and Laboratory Standards Institute released a protocol document relating to the controls. CLSI, a non-profit standards organization, made the controls available to parties that could not stand to wait several years to obtain the control information, such as diagnostic developers.
Last fall, the ERCC selected testing sites for the RNA controls, narrowing a field of candidate sites to Affymetrix, Agilent Technologies, Illumina, and the National Institute of Allergy and Infectious Diseases, which used a homebrew platform to test the controls (see BAN 10/3/2006).
Salit said last week that the first phase of testing at those sites was successful, and that it is likely that the ERCC will continue to refine its list of controls until the reference material is ready for distribution. NIST has yet to disclose a price tag for the plasmid library, although Salit estimated it could cost around $2,000.
“These are actual physical materials that can be used,” Salit said. “We will be offering plasmid in a tube. I would expect that the user of this reference material is going to be making RNA as control material from the plasmid library,” he said.
The RNA generated through the control sequences in the plasmid will be synthetic RNA that is intended to mimic mammalian messenger RNA, Salit said. “Most of them are random generated synthetic sequences that don’t Blast against any known genomes. There are also some bacterial sequences that have been in common use already as microarray controls, and then we have got some [infrequently occurring] extremophile sequences,” he added.
“The user is going to be a core lab or a commercial vendor that will make the RNA for sale as a control. I think it should support a given core lab’s need for a couple years at a time,” Salit added. He also said that an updated version of the CLSI document will probably be released after the controls become available sometime next year.
The MAQC and the ERCC
The ERCC project is part of a multitude of efforts across the microarray space to lend the technology, still considered cutting-edge and unreliable by some, some semblance of standardization. Another government-hosted undertaking that has gotten industry and academics to work together is the Microarray Quality Control project. Overseen by the US Food and Drug Administration, the MAQC published a series of papers in Nature Biotechnology last year detailing efforts to establish concordance among data generated across different microarray platforms and to calm reservations about the reproducibility of results gleaned from array experiments (see BAN 9/12/2006).

“I think it should support a given core lab’s need for a couple years at a time.”

More recently, the MAQC project has turned its attention to the predictive models that use gene expression data to predict disease recurrence, prognosis, response to treatment, and other outcomes of clinical interest (see BAN 6/5/2007).
Array vendors are still using the MAQC results to trumpet the strengths of their own platform. At the World Microarray Congress, held in Vancouver last month, several researchers used MAQC data in their presentations. But rather than use the ERCC solely as a marketing tool after it is released, Salit said last week that vendors will take an active role in integrating the ERCC reference material into their existing and future products.
“That’s the nice part about the ERCC; we are working with people that are developing the probe content as we speak so when the controls are available there will be catalog microarrays that can use them,” he said.
“They are designing probes on the arrays that will detect this kind of content for control purposes,” Salit said. “The cool thing is that you can add these to your study and you can be pretty confident that the controls you are adding aren’t going to cross-hybridize and give you false signals for the organism that you are looking at. Your organism of interest shouldn’t be interfered with by these controls,” he said.
Salit said that he expects considerable demand for the plasmid library, which will be manufactured by an undisclosed vendor. “I think that there’s a lot of enthusiasm in the community to get their hands on a sufficiently large library of common controls,” he said. “People are anxious for it.”
He added that other efforts, like the MAQC project, are “complementary where microarray measurement assurance is a primary concern. I think that in the end the whole community is going to strongly benefit from having [measurement assurance],” he said.

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