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Nucleic Acids Research Advance Access originally published online on September 25, 2006
Nucleic Acids Research 2006 34(18):e123; doi:10.1093/nar/gkl639
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Nucleic Acids Research, 2006, Vol. 34, No. 18 e123
© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Nanoliter high throughput quantitative PCR

Tom Morrison, James Hurley, Javier Garcia, Karl Yoder, Arrin Katz, Douglas Roberts, Jamie Cho, Tanya Kanigan, Sergey E. Ilyin1, Daniel Horowitz1, James M. Dixon1 and Colin J.H. Brenan*

BioTrove Inc. 12 Gill Street, Suite 4000, Woburn, MA 01810, USA 1 Johnson & Johnson Pharmaceutical Research & Development LLC, Spring House, PA 19477, USA

*To whom correspondence should be addressed. Tel: +1 781 721 3615; Fax: +1 781 721 3601; Email: cbrenan{at}biotrove.com

Received June 15, 2006. Revised August 16, 2006. Accepted August 16, 2006.

Understanding biological complexity arising from patterns of gene expression requires accurate and precise measurement of RNA levels across large numbers of genes simultaneously. Real time PCR (RT-PCR) in a microtiter plate is the preferred method for quantitative transcriptional analysis but scaling RT-PCR to higher throughputs in this fluidic format is intrinsically limited by cost and logistic considerations. Hybridization microarrays measure the transcription of many thousands of genes simultaneously yet are limited by low sensitivity, dynamic range, accuracy and sample throughput. The hybrid approach described here combines the superior accuracy, precision and dynamic range of RT-PCR with the parallelism of a microarray in an array of 3072 real time, 33 nl polymerase chain reactions (RT-PCRs) the size of a microscope slide. RT-PCR is demonstrated with an accuracy and precision equivalent to the same assay in a 384-well microplate but in a 64-fold smaller reaction volume, a 24-fold higher analytical throughput and a workflow compatible with standard microplate protocols.


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