| Gene Expression Analysis
Competitive PCR
Sequenom, Inc.
3595 John Hopkins Ct.
San Diego, CA 92121
aloria@sequenom.com
www.sequenom.com
858-202-9194
Quantitative Gene Expression Analysis with MassARRAY
®
System
MassARRAY
®
QGE is the ideal complement to fi ne 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-fl ight mass spectrometry
(MALDI-TOF MS). A protocol overview is provided in Figure 1.
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
Reverse
titration is set up to determine the competitor concentration at which
Transcription
RNA
amplifi cation between cDNA and competitor is equal (termed EC50).
During mass spectrometric analysis, the peak areas of the distinct
cDNA
mass signals for the competitor and cDNA extension products are
calculated.
Amplification
cDNA Competitor
C G
The QGE Analyzer software plots cDNA frequency versus competitor
cDNA amplicon Competitor amplicon
concentration for each assay and sample (shown below). cDNA
concentrations are automatically calculated via non-linear regression
C
G
G C
analysis and represent the competitor concentration at which the
allele frequencies of cDNA and competitor are equal.
Primer
MassARRAY
®
QGE offers cycle-independence, non-fl uorescent
Extension
G C
C G
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
Detection and
G
Ratio Analysis
employed, as little as 1 attomolar (~3 copies per reaction) can be C
detected.
References:
1. Ding C. and Cantor, C.R. PNAS USA. 2003 Mar 18: 100(6):3059-64.
Figure 1
2. Elvidge G.P., Price T.S., Glenny L., Ragoussis, J. Anal. Biochem. 2005
Apr 15: 339(2):231-41.
3. Turakulov, R. et al. Plant Cell Physiol. 48(9):1379–84 (2007).
4. Yang, H. et al. PNAS 2005 May 24: 102(21):7683–88.
Protocol Guide ı 2008 View entire protocol online at www.biotechniques.com/protocol ı BioTechniques ı 31
Seq GeneExp Protocol.indd 31 10/26/07 11:21:40 AM
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