Although PCR technology is nearly a quarter of a century old, scientists not only use it but depend on it, particularly in the areas of gene expression and microarray processes. Researchers discussed the latest advances in PCR at Cambridge Healthtech’s “Quantitative PCR” conference held recently in San Diego.
Microarrays are moving to the fore as the preferred way to perform parallel measurements of relative expression levels of large numbers of genes among different samples. One problem, however, is the fact that microarrays are notorious for suffering from systematic errors that diminish measured expression ratios below their true values. Agilent Technologies (www.agilent.com) has developed a way to use the combination of external spike-in RNA standards and a commercial direct mRNA assay (called the QuantiGene assay) to objectively optimize both microarray production methods and sample processing methods.
“It’s becoming popular and it’s about to become a lot more popular,” said Paul Wolber, integrating manager, microarray QC development at Agilent. “On September 10, a series of papers is going to be published in Nature Biotechnology that does a cross-platform comparison of all the major microarray and confirmation mRNA measurement technologies.””
Agilent is reversing the methods that pharmaceutical companies use to validate a particular target gene. “We’ve adopted a feedback method,” said Wolber. “If quantitative PCR (qPCR) methods are getting a result, we do two things to reduce the numbers of false positives and false negatives from an initial microarray screen.
“First, we validate the microarray results for certain genes via Quantigene under a variety of microarray processing conditions. Since Quantigene has been cross-validated with qPCR, this also serves to validate on that method.
“Next, we look at the behavior of a series of spiked-in mRNAs, which are basically a part of the gene,” said Wolber. “We use a set of targets derived from human adenovirus. We make mRNA synthetically in a traceable manner, then add known amounts of the various targets to real samples. Once again, we observe how close the microarray comes to delivering the known correct result for the spiked-in targets, under a variety of microarray processing protocols. The desired result is to optimize the protocol. The answer you get has to equal what you’ve put in.”
The optimization projects have resulted in substantial improvements in the accuracy of expression ratio measurements and demonstrate the power of orthogonal analytic methods to guide further advances in microarray technology. “There will be plenty of opportunities for mRNAs to help out PCRs,” Wolber added. “What we’re seeing is the co-evolution of two methodologies, where one can be used to help the other.”