Microarray-based, high-throughput gene expression profiling of microRNAs

Abstract

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.

Silicon nanowire-transistor biosensor for study of molecule-molecule interactions

“…The assay was further tested for the detection of miRNA in real samples by analyzing let-7b in total RNA extracted from HeLa cells. The concentration of let-7b detectable in the total RNA extracted from HeLa cells was 2.15 ± 0.25 × 107 copies/μg, which is in good agreement with the previously published data of miRNA expression profiling (Nelson et al 2004). This method shows potential applications in label-free, early detection of miRNA associated with cancer with high sensitivity and specificity.…”

MicroRNAs and their regulatory roles in animals and plants

“…miRNAspecific quantitive PCR and microarray technology make this study become easier. In the past 1 year, several quantitive PCR Raymond et al, 2005;Shi and Chiang, 2005) and microarray technology (Babak et al, 2004;Barad et al, 2004;Nelson et al, 2004b;Thomson et al, 2004;Liang et al, 2005) have been developed, and successfully used to study the functions of miRNAs in organism development (Miska et al, 2004), disease and cancer (Lu et al, 2005b). At present, several companies have developed miRNA chips for studying the expression profiles at a specific situation.…”

The role of microRNA in human leukemia: a review

“…28 The RNA primed, array-based Klenow enzyme assay uses on the slide application of the Klenow fragment of the DNA polymerase I to extend miRNAs hybridized to DNA probes. 29 Generation of small RNA libraries and deep sequencing using a high-throughput approach is another way of identification and relative quantification of miRNAs. 30 Each of these techniques has its strengths and weaknesses (Table 1), and the confirmatory use of a second independent technique is mandatory at the present time to identify false-positive/negative data for all the different assays undertaken.…”