After digestion, mouse and human cells were cultured overnight using MSFM or RSFM respectively in wells pre-coated with 0.1% gelatin. Media was supplemented with 20 ng/ml recombinant human GDNF, 150ng/ml recombinant rat GFR alpha 1 (R&D Systems Inc., Minneapolis, MN) and 1 ng/ml recombinant human bFGF (BD Biosciences cells were removed from the wells using gentle pipetting, then transferred into a 15 ml conical tube and collected by centrifugation at 4°C, 600 X g for 7 min. After washing with HBSS twice, the cell pellet was concentrated in MSFM or RSFM with GDNF, bFGF and GFR alpha 1. Cells were then placed in drops in a 10 cm culture dish and covered with silicone oil for micromanipulation.
Extracted molecule
total RNA
Extraction protocol
Microarray services were provided by the Penn Microarray Facility, including quality control tests of the total RNA samples by Agilent Bioanalyzer and Nanodrop spectrophotometry, as described in the NuGEN Ovation manual and the Affymetrix GeneChip Expression Analysis Technical Manual. Selected germ and somatic cells were placed in 25 ml Trizol reagent (Invitrogen) immediately after isolation
Label
biotin
Label protocol
Briefly, total RNA was converted to first-strand cDNA using reverse transcriptase primed by a poly(T) oligomer. Second-strand cDNA synthesis was followed by ribo-SPIA (Single Primer Isothermal Amplification, NuGEN Technologies Inc. San Carlo, CA) for linear amplification of each transcript, and the resulting cDNA was fragmented, assessed by Bioanalyzer, and biotinylated.
Hybridization protocol
cDNA were added to Affymetrix hybridization cocktails, heated at 99ºC for 2 min and hybridized for 16 h at 45ºC to Affymetrix Human Hu133+v2 and Mouse 430 2.0 GeneChips (Affymetrix Inc., Santa Clara CA). Each sample (n=6 per species) was hybridized to its own array.
Scan protocol
According to Affymetrix protocol
Description
Mouse_41
Data processing
Probe level data (.cel files) were created with Affymetrix Command Console and Expression Console software. They were imported into Partek Genomics Suite (version 6.4) were RMA was applied to calculate probe set intensities.
