BioProject

Microarray analysis of miRNA—Two micrograms of total RNA was extended at the 3′ terminus with a poly(A) tail using poly(A) polymerase. More...

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). Overall design: In the study presented here, compared the genome-wide expression patterns of miRNAs in MEFs, ESCs, and various iPSCs to identify miRNAs that were potentially involved in the LIF-independence. iPSCs in this study: O-retro-iPSCs-LIF(+), O-lenti-iPSCs-LIF(+), M3O-lenti-iPSCs-LIF(+), and M3O-lenti-iPSCs-LIF(-). O-retro-iPSCs-LIF(+) was established by transduction of the OSKM genes into MEFs with the pMXs-IP retroviral vector and maintained with LIF. O-lenti-iPSCs-LIF(+) was prepared with lentiviral Oct4 and retroviral Sox2, Klf4, and c-Myc (SKM) to maintain the expression of the Oct4 transgene in iPSCs as a control for the two M3O-based iPSC types. M3O-lenti-iPSCs-LIF(+) was the same as O-lenti-iPSCs-LIF(+) except that M3O was used instead of Oct4. M3O-lenti-iPSCs-LIF(-) was the same as M3O-lenti-iPSCs-LIF(+) except that LIF was omitted during establishment and maintenance. Less...