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Quantitative Gene Expression Analysis with MassARRAY® System
Sponsored,vendor-submitted protocol   Sponsored by Sequenom    Published in November 2007 (p.31)

MassARRAY® QGE is the ideal complement to fine mapping studies and solution for post-array validation. This methodology is orders of magnitude more sensitive than real-time quantitative PCR and permits very closely related genes to be assayed reliably and quantitatively. Independent studies show a high rate of concordance between MassARRAY® QGE, microarray data, and real-time PCR.

MassARRAY® QGE combines real-competitive PCR (rcPCR) with the iPLEXTM Gold primer extension reaction, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). A protocol overview is provided in Figure 1.

(Click to enlarge)

In the QGE method, a synthetic competitor serves as an internal standard to quantify gene expression levels. PCR primers bind and co-amplify the cDNA and competitor with equal stoichiometry and kinetics preserving the initial cDNA:competitor ratio. Since the cDNA concentration of any one transcript is unknown, a competitor titration is set up to determine the competitor concentration at which amplification between cDNA and competitor is equal (termed EC50). During mass spectrometric analysis, the peak areas of the distinct mass signals for the competitor and cDNA extension products are calculated.

The QGE Analyzer software plots cDNA frequency versus competitor concentration for each assay and sample (shown below). cDNA concentrations are automatically calculated via non-linear regression analysis and represent the competitor concentration at which the allele frequencies of cDNA and competitor are equal.

MassARRAY® QGE offers cycle-independence, non-fluorescent detection, and universal reaction conditions for all assays. Both relative and absolute expression levels can be measured using this method, and depending upon the range of the competitor titration employed, as little as 1 attomolar (˜3 copies per reaction) can be detected.

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