ES cells were trypsinized (d0) and cultured in suspension to form embryoid bodies (EBs). Briefly, ES cells were transferred into Iscove's modified Dulbecco's Medium (IMDM) with 20% batch-tested FCS, nonessential amino acids (0.1 mM) and beta-mercaptoethanol (0.1 mM) (Invitrogen, Karlsruhe, Germany) in a 10 cm bacterial Petri dish and cultured on a shaker for 48 h. At d2, EBs were transferred into spinner flasks (Integra Cell Spin, IBS, Fernwald, Germany) and cultured for additional 7 days at 37°C, 5% CO2, and 95% humidity. Medium was exchanged at d5, d7, and d9; and at day 9 Puromyicn was added to select for cardiomyocytes. At d12, remaining cardiobodies were trypsinized to obtain a single cell suspension of cardiomyocytes (Cor.At cardiomyocytes). Cor.At cells were seeded onto a Fibronectin coated 10cm dish as described above, and cultured for 6h before extracting RNA.
Growth protocol
aPIG 44 cells were cultured on mouse embroynic fibroblasts in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with nonessential amino acids (0.1 mM), L-glutamine (2 mM), beta-mercaptoethanol (0.1 mM), LIF (ESGR) (500 U/ml), neomycin (6 µg/mL), and batch-tested fetal calf serum (FCS) (15% v/v) (all Invitrogen, Karlsruhe, Germany).
Extracted molecule
total RNA
Extraction protocol
Total RNAs were extracted by using PeqGold RNApure (Peqlab Biotechnology, Erlangen, Germany) according to the manufacturer's instructions.
Label
biotin
Label protocol
Sample labeling with biotin has been carried out by microfluidic-based enzymatic on-chip labeling of miRNAs (MPEA).
Hybridization protocol
Hybridization for 16 hours at 42°C has been carried out overnight according to manufacturs instructions. Then, the biochip was washed automatically and a program for signal enhancement was processed with the Geniom Real Time Analyzer
Scan protocol
The resulting detection pictures were evaluated using the Geniom Wizard Software.
Description
mouse ES cells (D3, ATCC CRL 1934) transfected with the alpha-MHC-Pac-IRES-EGFP vector
Data processing
Data was normalized with VSN (Bioconductor, vsn package).
