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.

Discovery, evaluation, and understanding the biological relevance of single nucleotide polymorphisms (SNPs) and their associated phenotypes is relevant to many applications, including human disease diagnostics, pathogen detection, and identification of genetic traits impacting agricultural practices, both in terms of food quality and production efficiency. Validation of putative SNP associations in large-scale cohorts is currently impeded by the technical challenges and high cost inherent in analyzing large numbers of samples using available SNP genotyping platforms. We describe in this report the implementation of the 5'-exonuclease, biallelic PCR assay for SNP genotyping (TaqMan) in a nanofluidic version of a high-density microplate. System performance was assessed using a panel of 32 TaqMan SNP genotyping assays targeted to human polymorphisms. This functional test of the nanoliter fluidic SNP genotyping platform delivered genotyping call rates and accuracies comparable to the same larger volume reactions in microplate systems.