Nanoliter high throughput quantitative PCR.
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.
Morrison
Tom
T
BioTrove Inc, 12 Gill Street, Suite 4000, Woburn, MA 01810, USA.
Hurley
James
J
Garcia
Javier
J
Yoder
Karl
K
Katz
Arrin
A
Roberts
Douglas
D
Cho
Jamie
J
Kanigan
Tanya
T
Ilyin
Sergey E
SE
Horowitz
Daniel
D
Dixon
James M
JM
Brenan
Colin J H
CJ
D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D008099:Liver / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010770:Phosphotransferases / Q000096:biosynthesis / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000378:metabolism; D015203:Reproducibility of Results; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D010770:Phosphotransferases