Application of the primRglo assay chemistry to multiplexed bead assays

Fang, Liang, Weis, Anna, Wong, Lawrence Kurniawan, Yeh, David Che-Cheng, Lai, Richard, Corrie, Simon and Barnard, Ross T. (2014) Application of the primRglo assay chemistry to multiplexed bead assays. Current Protocols in Cytometry, 69 13.13.1-13.13.10. doi:10.1002/0471142956.cy1313s69

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Author Fang, Liang
Weis, Anna
Wong, Lawrence Kurniawan
Yeh, David Che-Cheng
Lai, Richard
Corrie, Simon
Barnard, Ross T.
Title Application of the primRglo assay chemistry to multiplexed bead assays
Journal name Current Protocols in Cytometry   Check publisher's open access policy
ISSN 1934-9297
ISBN 9780471142959
Publication date 2014-07-01
Year available 2014
Sub-type Article (original research)
DOI 10.1002/0471142956.cy1313s69
Open Access Status
Volume 69
Start page 13.13.1
End page 13.13.10
Total pages 10
Publisher John Wiley and Sons
Collection year 2015
Language eng
Abstract In this unit, we describe a multiplex microsphere quantitative PCR. The system is based on the use of two additional oligonucleotides within a single tube PCR reaction. The first oligonucleotide is modified with a single base pair mismatch and is otherwise equivalent to a universal sequence added to the forward PCR primer. Further, this first extra oligonucleotide is coupled to Luminex microspheres. The second additional oligonucleotide is designed to be complementary to the universal sequence, and is modified with the fluorescent dye Cy3. As the PCR reaction proceeds, the second oligonucleotide is able to bind to the microspheres. Thus, quantitative monitoring of PCR progress takes place. The microsphere-mediated Cy3-detection is measured using flow cytometry directly after the PCR reaction. This allows a flow cytometer analysis from up to 150 different spheres and, therefore, multiple genes in one reaction. The multiplex microsphere qPCR is demonstrated using three target genes from Influenza A and Neisseria meningitidis. The multiplex microsphere system will enable a higher degree of multiplexing than is possible with currently available qPCR systems.
Keyword Flow cytometry
Q-Index Code CX
Q-Index Status Confirmed Code
Institutional Status UQ
Additional Notes Unit 13.13

Document type: Journal Article
Sub-type: Article (original research)
Collections: Non HERDC
School of Chemistry and Molecular Biosciences
Australian Institute for Bioengineering and Nanotechnology Publications
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Created: Fri, 11 Jul 2014, 10:22:56 EST by Mrs Louise Nimwegen on behalf of School of Chemistry & Molecular Biosciences