normalization data transformation protocol
The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 100. ID_REF = VALUE = Signal ABS_CALL = indicating whether the transcript was present (P), absent (A), or marginal (M) DETECTION P-VALUE =
array scanning protocol
GeneChips were scanned using theAffymetrix GCS3000 scanner.
Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Mouse MU74Av2 Arrays. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 5 ug total RNA.
nucleic acid extraction protocol
Total RNA was harvested from perifused cells on cytodex 3 microcarriers using Trizol LS and purified on RNeasy spin columns (Qiagen, Valencia, CA). The mRNA was quantified and its integrity checked by agarose gel electrophoresis.
LbT2 cells were maintained in monolayer cultures in DMEM supplemented with 10% fetal bovine serum and antibiotics in a humidified 10% CO2 atmosphere at 37 °C. For perfusion studies, cells were plated on cytodex 3 microcarrier beads (GE Healthcare, Buckinghamshire, England) at a density of 1.5 x 107 cells/ml bed volume. After culture for 5 days, cells were pelleted by spinning at 1000Xg for 1 minute, washed once with Serum-Free DMEM, re-pelleted, and placed in Serum-Free DMEM 16-18h.
sample treatment protocol
Cells were then loaded into perifusion columns and equilibrated for 1 h in serum-free DMEM supplemented with penicillin and streptomycin at a flow rate of 300 microliters/min. Subsequently, cells were pulsed for 2 minutes once or at 28, 58, 118 minute intervals or given tonic 10 or 100 nM GnRH for 4 h for the microarray studies, or 6h for the transfection studies. Perifusion chambers were constructed from C10 chromatography columns (GE Healthcare, Buckinghamshire, England) with modified flow adapters and cells were maintained by perifusion with DMEM equilibrated in an atmosphere of 5% CO2 using a minipulse 3 peristaltic pump (Gilson, Middleton, WI).