Stage 7 embryos were treated in groups of 25 in 60-mm Petri dishes with 10 mL of 0.1x MBS containing 10-6M RAR agonist (TTNPB), 10-6M RAR inverse agonist (AGN193109), or vehicle control (0.1% EtOH). Three dishes, per treatment, per female were collected (27 dishes total: 3 technical replicates, 3 biological replicates per treatment).
Growth protocol
Xenopus laevis eggs from three different females were fertilized in vitro and embryos were staged according to Nieuwkoop and Faber
Extracted molecule
total RNA
Extraction protocol
Each dish of embryos was harvested at stage 18 into 1.5 mL of RNAlater (Invitrogen) and kept overnight at 4C and until use. RNAlater was replaced in the 2 ml plastic tube with 1.0 ml of RLT buffer (Qiagen GmbH., Germany), and homogenized by adding a 5 mm diameter Zirconium bead (Funakoshi, Japan) and shaking with a MixerMill 300 (Qiagen GmbH., Germany) at a speed of 20Hz for 5 min (only the outermost row of the shaker box was used). Three separate 10μl aliquots were taken from each sample homogenate to another tube and mixed thoroughly. A final 10 μl aliquot there from was treated with DNase-free RNase A (Nippon Gene Inc., Japan) for 30 min at 37C, followed by Proteinase K (Roche Diagnostics GmbH., Germany) for 3 h at 55C in 1.5 ml capped tubes. The aliquot was transferred to a 96-well black plate. PicoGreen fluorescent dye (Molecular Probes Inc., USA) was added to each well, shaken for 10 seconds four times and then incubated for 2 min at 30C. The DNA concentration was measured using a 96 well fluorescence plate reader with excitation at 485nm and emission at 538nm. λ phage DNA (PicoGreen Kit, Molecular Probes Inc., USA) was used as standard. As reported previously, the grade-dosed spike cocktail (GSC) made of the following five Bacillus subtilis RNA sequences were selected from the gene list of Affymetrix GeneChip arrays (AFFX-ThrX-3_at, AFFX-LysX-3_at, AFFX-PheX-3_at, AFFX-DapX-3_at, and AFFX-TrpnX-3_at) present in the arrays was added to the sample homogenates in proportion to their DNA concentrations (BMC Genomics 7 64 2006). Then, the sample homogenates spiked with GSC were processed by RNeasy Mini Kit (Qiagen GmbH., Germany).
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the manufacturer's instructions from 5 ug total RNA by Enzo BioArray High Yield RNA Transcript Labeling Kit (Enzo Life Sciences) .
Hybridization protocol
Following fragmentation, 15 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Xenopus laevis Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
Scan protocol
GeneChips were scanned using the Affymetrix GeneChip Scanner 3000.
Description
percellome normalization (absolutized mRNA copy numbers per cell, see BMC Genomics 7 64 2006) CPC-G, 109
Data processing
The raw data were summarized by Affymetrix MAS5. The efficiency of in vitro transcription (IVT), and the dose-response linearity of the five GSC spikes was checked, and Percellome data, i.e. absolutized mRNA copy numbers of each ProbesSet were generated by the homemade software SCal4 (Spike Calculation version 4, by Ken-ichi Aisaki) Percellome microarray data (copies per cell) were analyzed using CyberT statistical analysis, which corrects for low replicates and variability, allowing for less false negatives under two-fold change, and less false positives for large fold changes (Arima et al., 2005; Baldi and Long, 2001; Kayala and Baldi, 2012).
