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Genome-wide identification and analysis of microRNA expression in MEFs, ESCs, and iPSCs

ABSTRACT: Microarray analysis of miRNA—Two micrograms of total RNA was extended at the 3′ terminus with a poly(A) tail using poly(A) polymerase. An oligonucleotide tag was then ligated to the poly(A) tail for later fluorescent dye staining. Hybridization was performed overnight on a µParaflo microfluidic chip using a microcirculation pump (Atactic Technologies). On the microfluidic chip, each detection probe consisted of a chemically modified nucleotide coding segment complementary to the target microRNA (miRBase, http://mirbase.org) and a spacer segment of polyethylene glycol to extend the coding segment away from the substrate. The detection probes were made by in situ synthesis using photogenerated reagent chemistry. The hybridization melting temperatures were balanced by chemical modifications of the detection probes. Hybridization used 100 µl 6× SSPE buffer (0.90 M NaCl, 60 mM Na2HPO4, 6 mM EDTA, pH 6.8) containing 25% formamide at 34°C. After RNA hybridization, tag-conjugating Cy5 dye was circulated through the microfluidic chip for dye staining. Fluorescence images were collected by using a laser scanner (GenePix 4000B, Molecular Device) and digitized using Array-Pro image analysis software (Media Cybernetics). Data were analyzed by first subtracting the background and then normalizing the signals using a LOWESS filter (Locally-weighted Regression).

ORGANISM(S): Mus musculus

PROVIDER: GSE65596 | GEO | 2015/10/01

SECONDARY ACCESSION(S): PRJNA274448

REPOSITORIES: GEO

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Genome-wide identification and analysis of microRNA expression in MEFs, ESCs, and iPSCs

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