P19C6, a subclone of the mouse EC P19 cell line 4 day treatment
Treatment protocol
For N2B27 induction,nearly confluent P19 cells were dissociated into single cells by Trypsin-EDTA ,and allowed to aggregate in the bacteriological petri dishes at a seeding density of 1 × 105/ml in N2B27 medium (1:1 mix of DMEM/F12 supplemented with 25 μg/ml insulin, 100 μg/ml apo-transferrin, 6 ng/ml progesterone, 16 μg/ml putrescine, 30 nM sodium selenite, 50 μg/ml bovine serum albumin fraction V, and neurobasal medium supplemented with B27 without Vitamin A). After 4 days of culture in suspension, the cell aggregates were collected, rinsed once with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde for 30 min, incubated in 20% sucrose overnight, embedded in cryomedium OCT,cryo-sectioned at a thickness of 15 μm, and stored at −20°C for further use. The factors and inhibitors, such as bone morphogenetic protein 4 (BMP4) (10 ng/ml), Noggin (100 ng/ml), SU5402 (5 μM), U0126 (5 μM), Wortmannin (100 nM), or U73122 (100 nM), were added at the beginning of the suspension culture, and were present throughout the experiments. To caudalize the N2B27-induced neural stem cells, the RA (2 μM) was added to the N2B27 medium for another 2–4 days, and the cell aggregates cultured in N2B27 medium for 6–8 days served as the control.
Extracted molecule
total RNA
Extraction protocol
The total RNAs were extracted with the RNarose reagent (Watson and Sangon).
Label
Cy5
Label protocol
labeled by PCR using a Cy5 labeled primer
Hybridization protocol
Microscope glass slides were activated as described.The antisense sequences of microRNAs were used for probes. The melting temperatures of all probes were adjusted to 55 ± 3 °C as described.In addition, each probe contains a 5′ C6-amino modified 10 deoxyadenosines spacer at the 5′ end of the probe.The probes were dissolved in printing buffer (2× SSC/1 M betaine hydrochloride/5% formamide/0.01% SDS, pH 8.5) at the concentration of 20 μM, and then printed onto the epoxy coated slides with PixSys 5500 spotting robot in quadruplicate under 60% humidity. The printed microarrays were further chemically covalently coupled under 60% humidity overnight. Before hybridization, printed microarrays were quenched by quenching buffer (0.1 M Tris–HCl/50 mM ethanolamine, pH 8.0). Small RNA cDNA libraries of samples were amplified and labeled by PCR using the 5′primer (5′-Cy5-CTGGAATTCTGACGATCTGC) and the 3′primer (5′-ACCGAATTCACAGTCAGACC) as described [11]. The PCR product was precipitated, washed, resuspended in 10 μl ddH2O, and cleaned up by a Sephadex G-50 column. Heat the PCR product at 95 °C for 3 min to denature and chill on ice immediately. Combine the PCR product with equal volume of 2× hybridization buffer (8× SSC/0.2% SDS, pH 7.0) that has been heated to 48 °C and mix thoroughly. Immediately, inject the mixture into the hybridization chamber. Hybridization was carried out at 45 °C for 16 h. After hybridization, slides were washed (2× SSC/0.03% SDS, 2 min; 0.2× SSC, 2 min; 0.1× SSC, 1 min) at 25 °C.
Scan protocol
scaned by ScanArray5000
Description
analysis of mouse microRNAs expression under neural differentiation
Data processing
Signal intensities for each spot were calculated by subtracting local background from total intensities. No further process was performed.
