7 protocols
AccessionType
bioassay_data_transformation
Microarray data extraction, normalization and data analysis were carried out as described recently by Pothof J, Verkaik NS, van Ijcken W, Wiemer EA, Ta VT, et al. (2009) MicroRNA-mediated gene silencing modulates the UV-induced DNA-damage response. EMBO J.

experimental groups after normalisation and retransformation(2log) to normal values

mean+sd of precursor cells (elg13+elg14+elg15)

mean+sd of immature DC (elg16+elg17+elg18)

mean+sd of mature DC (elg19+elg20+elg21)

mean+sd of LPS stimulated mature DC (elg22+elg23+elg24)
bioassay_data_transformation
Microarray data extraction, normalization and data analysis were carried out as described recently by Pothof J, Verkaik NS, van Ijcken W, Wiemer EA, Ta VT, et al. (2009) MicroRNA-mediated gene silencing modulates the UV-induced DNA-damage response. EMBO J.
image_acquisition
Slides were washed and immediately scanned using a Tecan LS Reloaded microarray laser scanner.
(Parameters: Scanning hardware = Tecan LS, Scanning software = Scanning software)
labeling
RNA was labelled using a ULS™ aRNA labeling kit (Kreatech Diagnostics, Amsterdam). 1.5 ug of total RNA was incubated with Cy3-ULS for 30 min at 85 °C and purified to remove unbound Cy3-ULS.
(Parameters: Amount of nucleic acid labeled = 1.5, Amplification = none, Mass unit = Micro gram)
grow
Bone marrow cells isolated from 8-13 wk old C57BL/6 mice were cultured in RPMI-1640 medium (Lonza, Belgium) supplemented with 10% fetal calf serum, 2 mM glutamine, 100 U/ml penicillin, 100 ug/ml streptomycin, 50 uM 2-mercaptoethanol and 20 ng/ml rmGM-CSF (Biosource International, Camarillo, CA). Cells were kept in a humidified incubator at 37°C with 5% CO2. At day 0, BM leukocytes were seeded at 3×10e5 per ml in either 100 mm dishes (BD Biosciences), 12-wells plates (Nunc) or 96-wells round-bottom plates (Nunc). At day 3, fresh culture medium was added to the plates and at day 6, half of the medium was replaced. To induce enforced DC maturation, 100 ng/ml LPS (Escherichia coli strain 055:B5, Sigma) was added on day 6. Cells were sorted on d8 according to CD11c, CD86 and MHC class II expression into 3 populations; precursors, immature DC (iDC) and mature DC (mDC).
(Parameters:mass unit = micro gram. time unit = days, temperature unit = C, media = RPMI1640)
nucleic_acid_extraction
Total RNA was extracted using acid-phenol:chloroform (Ambion) extraction and enriched for microRNAs using a mirVana microRNA isolation kit (Ambion) according to the manufacturer’s protocols.
(Parameters: Extracted product = total_RNA, Amplification = none)
hybridization
Labelled RNA was hybridized on miRCURY LNA microRNA arrays (probe set 8.0; Exiqon, Vedbaek, Denmark) at 60 °C for 16h using a Tecan 4800 hybridization station
(Parameters: Chamber type = Tecan 4800, Quantity of label target used = 1.5, Mass unit = Micro gram, time = 16, Tiny time unit = hours, Volume = 105, Volume unit = Micro litre, temperature = 60)