Abstract
Microarray technology has become a very popular approach in cases where multiple experiments need to be conducted repeatedly or done with a variety of samples. In our lab, we are applying our high density spots microarray approach to microscopy visualization of the effects of transiently introduced siRNA or cDNA on cellular morphology or phenotype. In this publication, we are discussing the possibility of using this micro-scale high throughput process to study the role of microRNAs in the biology of selected cellular models. After reverse-transfection of microRNAs and siRNA, the cellular phenotype generated by microRNAs regulated NF-κB expression comparably to the siRNA. The ability to print microRNA molecules for reverse transfection into cells is opening up the wide horizon for the phenotypic high content screening of microRNA libraries using cellular disease models.
Highlights
Two key events in biological sciences happened a decade ago around the year 2000: first, the sequence of the human genome was completed and published by Celera Co. and NIH, and second, the discovery of RNA interference was reported, where small double-stranded oligonucleotides mediateMicroarrays 2013, 2 post-transcriptional gene silencing in a broad variety of organisms [1,2,3]
For the siRNA experiment, we selected OTP smart pool siRNA designed against the regulation of NF-κB (RelA) member of the NF-κB family and, as a negative control, the OTP non-targeted siRNA #1
As any pooled siRNA, this sample consists of four different siRNA sequences against RelA to increase the silencing efficiency and decrease the chance of off-target effects
Summary
Two key events in biological sciences happened a decade ago around the year 2000: first, the sequence of the human genome was completed and published by Celera Co. and NIH, and second, the discovery of RNA interference was reported, where small double-stranded oligonucleotides mediateMicroarrays 2013, 2 post-transcriptional gene silencing in a broad variety of organisms [1,2,3]. Double-stranded RNA was found to mediate sequence-specific, post-transcriptional knock-down of the mRNA in cells and tissues of all origins of plants, insects and animals. RNA-mediated interference (RNAi) became useful in the analysis of functional genes and their roles in biological phenotypes in mammalian and particular human cells [3,4]. The miRNAs (microRNA, miR) in contrast to the synthetic inhibitory RNAs, like siRNA and shRNA, are encoded by the genome and function as endogenous regulatory factors for both protein coding genes and non-coding RNAs. The expression of specific miRNAs in mammalian tissue was found to negatively correlate with the level of the mRNAs for genes whose 3'UTRs are targeted by miRNA [8]. MiRNA can induce two processes: the more common pathway involves promoting mRNA degradation by creating a perfect duplex and, inducing duplex cleavage by the RNA-induced silencing complex (RISC)
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