PCR and qPCR
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qPCR validation of microarray data using PrimeTime® qPCR Assays and ZEN™ Double-Quenched Probes

Tsai S, Hardison NE, et al. (2011) Transcriptional profiling of human placentas from pregnancies complicated by preeclampsia reveals disregulation of sialic acid acetylesterase and immune signaling pathways. Placenta, 32(2):175–182.

Preeclampsia, characterized by de novo onset of maternal hypertension and proteinuria, is a multi-system pregnancy-associated disorder that increases the risk of perinatal morbidity and mortality. This disorder is triggered by endothelial cell dysfunction that can arise from placental factors. In this study, Tsai et al. used genome-wide expression profiling of 60 human placentas to uncover gene expression patterns associated with preeclampsia. To validate the microarray data, the researchers performed qRT-PCR using PrimeTime® qPCR Assays from IDT. Their assays incorporated ZEN™ Double-Quenched Probes, which allow higher sensitivity over traditional single-quenched probes. The data revealed upregulation of sialic acid acetylesterase and sialic acid binding Ig-like lectin 6 and downregulation of ST6 β-galactosamide α-2,6-sialyltransferase 1 in the preeclamptic placenta.

Product focus: qPCR Reagents—everything but your sample

All the reagents you need for successful qPCR assays are available through IDT.

Double-Quenched Probes

ZEN™ and TAO™ Double-Quenched Probes have a 5′ fluorophore, an internal quencher (ZEN or TAO quencher), and Iowa Black FQ as the 3′ quencher. These probes provide consistently earlier Cq values and improved precision, when compared to traditional, single-quenched qPCR probes.

Learn more at www.idtdna.com/qPCRprobes.

Related reading

Decrease qPCR background, improve qPCR signal—Review data demonstrating that probes containing a second, internal quencher provide increased signal detection and greater assay sensitivity in qPCR assays vs. single-quenched probes, such as probes with BHQ Quenchers. ZEN™ and TAO™ Double-Quenched Probes also make it more feasible to use longer probes due to the resulting lower background fluorescence.

Increasing ddPCR performance in low target HIV assays—ZEN™ Double-Quenched qPCR probes work well in ddPCR. Learn how Dr Matthew Strain’s lab used ZEN™ Double-Quenched qPCR Probes in ddPCR, to achieve additional sensitivity through lower background in experiments with low copy number samples, where individual droplets matter. The Strain lab has published open-access protocols for their HIV assays that include these updated ZEN probe designs at www.bioprotocol.org.

Strategies for optimizing high throughput qPCR for expression profiling—Webinar summary: Find out how to address the challenges of high throughput RT-qPCR expression profiling from a prominent qPCR expert, Dr. Mikael Kubista (TATAA Biocenter, Sweden).

Review other DECODED Online newsletter articles on PCR and qPCR applications.

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Author: Ellen Prediger, PhD, is a senior scientific writer at IDT.

© 2013, 2016 Integrated DNA Technologies. All rights reserved. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners. For specific trademark and licensing information, see www.idtdna.com/trademarks.

Predesigned qPCR Assays

Probe-based qPCR assays for quantification of human, mouse, and rat gene expression. Order in plates or tubes.

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