Microarray-based, high-throughput gene expression profiling of microRNAs.
MicroRNAs (miRNAs) are small regulatory RNAs that serve fundamental biological roles across eukaryotic species. We describe a new method for high-throughput miRNA detection. The technique is termed the RNA-primed, array-based Klenow enzyme (RAKE) assay, because it involves on-slide application of the Klenow fragment of DNA polymerase I to extend unmodified miRNAs hybridized to immobilized DNA probes. We used RAKE to study human cell lines and brain tumors. We show that the RAKE assay is sensitive and specific for miRNAs and is ideally suited for rapid expression profiling of all known miRNAs. RAKE offers unique advantages for specificity over northern blots or other microarray-based expression profiling platforms. Furthermore, we demonstrate that miRNAs can be isolated and profiled from formalin-fixed paraffin-embedded tissue, which opens up new opportunities for analyses of small RNAs from archival human tissue. The RAKE assay is theoretically versatile and may be used for other applications, such as viral gene profiling.
Nelson
Peter T
PT
Department of Pathology and Laboratory Medicine, School of Medicine, 422 Curie Blvd., University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Baldwin
Don A
DA
Scearce
L Marie
LM
Oberholtzer
J Carl
JC
Tobias
John W
JW
Mourelatos
Zissimos
Z
D002460:Cell Line; D004256:DNA Polymerase I / Q000737:chemistry* / Q000235:genetics*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D020869:Gene Expression Profiling / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D035683:MicroRNAs / Q000032:analysis / Q000737:chemistry* / Q000235:genetics*; D046228:Microarray Analysis / Q000295:instrumentation / Q000379:methods*
D035683:MicroRNAs; D004256:DNA Polymerase I