Abstract

One-step
reverse transcription-PCR (RT-PCR) is widely used for gene expression analysis,
RNA virus detection, and routine RNA quantification experiments.

In this
study, we evaluated the IntelliQube^® real-time PCR instrument for
multiplex gene expression analysis using commercially available RNA from human
liver, lung, and kidney tissue, a one-step RT-PCR master mix, and BHQ^®
probe-based assays from LGC Biosearch Technologies. BHQ probes and primers were
designed to target the mucin 1 (MUC1), glyceraldehyde-3-phosphate dehydrogenase
(GAPDH), and the TATA-box binding protein (TBP) in a triplex PCR reaction. The
IntelliQube utilizes the Array Tape^® consumable and combines liquid
handling with real-time qPCR analysis in 1.6 µL reaction volumes.

Using TBP
and GAPDH as reference genes, it was determined that Muc1 was upregulated in
kidney 143-fold and lung 297-fold in comparison to liver, which correlated with
previously published data. Using a three way ANOVA, it was shown that there was
no significant difference in results between singleplex and triplex reaction
formats, or between multiple runs on the IntelliQube.

This study
demonstrates the ability of this instrument and the associated Array Tape
technology to successfully multiplex BHQ qPCR assays in a one-step RT-PCR
format for gene expression studies, without compromising data quality. Combined
with the automated inline process and economic benefits of Array Tape, the
IntelliQube and associated BHQ probe chemistry may prove to be a very useful platform
for multiplex qPCR applications in Human Genetics.