RT4 cell line is a urinary bladder transitional cell papilloma cell line from the American Type Culture Collection, Manassas Virginia.
Biomaterial provider
American Type Culture Collection, Manassas Virginia
Treatment protocol
Three dimensional gel cultures with Matrigel were performed by layering 0.8 mls of ice cold Matrigel onto polyethylene terephthalate membranes of 6-well cell culture inserts (Falcon, Becton-Dickinson Labware, Franklin Lakes, NJ). Gels were solidified at 37º C. RT4 cells were trypsinized, brought up on 20 mls McCoys media with 10% Fetal Calf Serum (FCS). Cells were centrifuged then brought up in McCoys media with 10% FCS to a concentration of 500,000 cells/250µl. 250µl added on top of each solidified matrigel disc and 2mls of McCoys with 10% FCS layered underneath each culture insert
Growth protocol
Cells were maintained for 10 days and media replenished underneath the insert twice a week. Cells were harvested at 12 hrs, 24hrs, 2 days, 3 days, 4days, 6 days, 7 days, 8 days, and 9 days of growth in culture using Matrisperse (Falcon, Becton-Dickinson Labware, Franklin Lakes, NJ) and RNA collected
Extracted molecule
total RNA
Extraction protocol
RNA was isolated at each time point with RNAeasy kit (QIAGEN Inc.,Valencia, CA) according to manufacturer’s protocol
Label
Cy3
Label protocol
Omniscript reverse-transcriptase, 37 C, 2 hrs
Hybridization protocol
Ventana Discovery 9h, 58 C washed to 0.1xSSC
Scan protocol
scanned on Agilient G2505B scanner Imagene 5.0
Description
Details about microarray handling/processing techniques can be obtained from platform’s manufacturer
Data processing
Data were normalized as was described previously in Dozmorov I et al., Nucleic Acids Res. 2004 Oct 28;32(19). In general, intensities were converted to expression and normalized to low-expressed genes, which provide possible technical noise level. The arrays are then adjusted to each other by robust linear regression and this Log10 transformed data presented for each sample. Group of low-expressed genes is selected by normal distribution fitting. This group provides internal standard of measurement noise (ISMN). Genes above 5SD from mean of ISMN were selected as expressed
