title: Wilms2_rep3

description: US22502668_251485010290_S01_GE1-v5_91_0806_1_3.txt

source_name: Wilms tumor cell line 2 replicate 3


title: Wilms1_rep5

status: Public on Feb 01 2010

type: RNA

city: Duesseldorf

first: Brigitte

last: Royer-Pokora

address: Universitätsstr. 1

country: Germany

institute: Institute of Human Genetics and Anthropology, Heinrich-Heine-University Duesseldorf

description: US45103065_251485038458_S01_GE1-v5_95_Feb07_1_4.txt

hyb_protocol: 1.65 ug of Cy3-labeled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 µl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 µl of 2x GE hybridization buffer HI- RPM was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (4x 44K) for 17 hours at 65°C in a rotating Agilent hybridization oven.  After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent) and air dried.

scan_protocol: Slides were scanned with an Agilent Technologies Scanner G2505B using a scan area of 61x21.6 mm, scan resolution of 5µm, dye channel set to green and green PMT set to 100%.

data_processing: Scans were quantified using Agilent Feature Extraction V10.1.1.1 Software using default parameters (protocols GE1-V5_91 and GE1-V5_95, as indicated by raw data file names). Control spots are removed, replicate oligos are averaged.

molecule: total RNA

organism: Homo sapiens

source_name: Wilms tumor cell line 1 replicate 5

label_protocol: Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN, Valencia, CA).  Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.

growth_protocol: For all Wilms (primary Wilms tumor cultures from different patients) and MSC samples: cells were cultivated in MSCB-medium supplemented with Bullet Kit (Lonza). For CLS1 samples: cells were cultivated in Iscoves medium supplemented with 2mM L-Glutamine and 15% FCS. The cultures were incubated at 37 ºC in a humidified incubator with 5% CO2.

extract_protocol: RNA was prepared using the RNAeasy Mini Kit (QIAGEN, Valencia, CA) following the manufacturer's recommendation with an included on-column DNase digestion. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).

tag: tissue

value: kidney

tag: gender

value: female

tag: age

value: 12 month

tag: cell type

value: Wilms tumor cell line

tag: genome/variation

value: WT1/CTNNB1 mut