Quantitative PCR based expression analysis on a nanoliter scale using polymer nano-well chips.
The analysis of gene expression is an essential element of functional genomics. Expression analysis is mainly based on DNA microarrays due to highly parallel readout and high throughput. Quantitative PCR (qPCR) based expression profiling is the gold standard for the precise monitoring of selected genes, and therefore used for validation of microarray data. Doing qPCR-based expression analysis in an array-like format can combine the higher sensitivity and accuracy of the qPCR methodology with a high data density at relatively low costs. This paper describes the development of an open-well based miniaturized platform for liquid PCR-based assays on the nanoliter scale using cost-effective polypropylene micro reactors (microPCR Chip). We show the quantification ability and reliability of qPCR in 200 nl with the microPCR chip down to 5 starting target molecules using TaqMan chemistry. An RNA expression analysis of four genes in mouse brain, liver and kidney tissues showed similar results in 200 nl as compared to standard 10 microl assays. The high sensitivity and quantification capability of the microPCR chip platform developed herein makes it a promising technology for performing high-throughput qPCR-based analysis in the nanoliter volume range.
Dahl
Andreas
A
Max-Planck-Institute for Molecular Genetics, Department Vertebrate Genomics, Ihnestrasse 73, 14195 Berlin-Dahlem, Germany. dahl@molgen.mpg.de
Sultan
Marc
M
Jung
Alexander
A
Schwartz
Regine
R
Lange
Matthias
M
Steinwand
Michael
M
Livak
Kenneth J
KJ
Lehrach
Hans
H
Nyarsik
Lajos
L
D000818:Animals; D020869:Gene Expression Profiling / Q000295:instrumentation* / Q000379:methods; D051379:Mice; D046228:Microarray Analysis / Q000295:instrumentation* / Q000379:methods; D008832:Microchemistry / Q000295:instrumentation* / Q000379:methods; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods; D036103:Nanotechnology / Q000295:instrumentation* / Q000379:methods; D016133:Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity