High-Throughput and Complex Gene Expression ValidationUsing the Universal ProbeLibrary and the LightCyclerR 480 System

摘要

During the past decade, a considerable number of transcrip-tome profiling analyses have been reported. High-through-put approaches such as tagging-based serial analysis ofgene expression (SAGE) and hybridization-based microar-ray technologies have been widely performed to profile geneexpression [1]. However, these techniques are either labori-ous or costly, or may involve advanced technologies whichlimit the sampling depth, thus diminishing the sensitivity ofanalysis. Validation of gene expression profiles can be rapidlyperformed using an independent technique such as quan-titative polymerase chain reaction (qPCR) to eliminate falseresults. In most laboratories, qPCR analysis has so far beenperformed on a small scale. This limits reliable downstreamanalysis of molecular interactions and pathway predictions,which are becoming important in the emerging field of sys-tems biology. qPCR study design involves consideration oftemplate quality and quantity, various levels of normalization,PCR efficiency (specificity and sensitivity), and data analysis.These factors are vital to ensure a reliable final quantifica-tion. The qPCR design becomes difficult when these factorsare considered for a study involving multiple tissues, ages,genes, groups (e.g., treatment and control groups) and bio-logical subjects. In this article, we discuss how the UniversalProbeLibrary and LightCycler R 480 System provided flexibil-ity in high-throughput confirmatory qPCR analysis of data-sets generated from SAGE and microarray platforms in threecomplex studies (Table 1).