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Sample GSM1380852 Query DataSets for GSM1380852
Status Public on May 06, 2014
Title Copies per Cell, Frog G, AGN193109
Sample type RNA
 
Source name Xenopus embryos treated with AGN193109 before gastrulation, collected at stage 18
Organism Xenopus laevis
Characteristics tissue: whole embryo
Stage: Nieuwkoop & Faber St. 18
agent: AGN193109
Treatment protocol 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).
 
Submission date May 06, 2014
Last update date May 06, 2014
Contact name Stefan Heller
E-mail(s) hellers@stanford.edu
Organization name Stanford University
Department Otolaryngology
Street address 801 Welch Road
City Stanford
State/province CA
ZIP/Postal code 94304
Country USA
 
Platform ID GPL1318
Series (1)
GSE57352 Expression data from Xenopus embryos treated with TTNPB (RAR-agonist), AGN193109 (RAR-antagonist), or Control Vehicle

Data table header descriptions
ID_REF
VALUE Percellome absolutized data (absolutized mRNA copy numbers per cell, see BMC Genomics 7 64 2006).

Data table
ID_REF VALUE
Xl.1.1.S1_s_at 217.6104
Xl.10.1.S1_s_at 2.2649
Xl.100.1.S1_at 27.5085
Xl.1000.1.S1_at 2.3500
Xl.10000.1.A1_at 18.2320
Xl.10001.1.A1_at 2.0740
Xl.10002.1.A1_at 6.8615
Xl.10004.1.A1_at 10.4130
Xl.10004.3.S1_a_at 6.6558
Xl.10006.1.A1_at 21.1821
Xl.1001.1.S1_at 1.4407
Xl.10011.1.A1_at 16.6288
Xl.10012.1.A1_at 23.1519
Xl.10013.1.A1_at 7.5893
Xl.10016.1.A1_at 36.7686
Xl.10019.1.A1_at 14.9163
Xl.1002.1.S1_at 10.3024
Xl.10021.1.A1_at 0.7779
Xl.10023.1.A1_at 0.8229
Xl.10024.1.S1_at 9.5301

Total number of rows: 15610

Table truncated, full table size 368 Kbytes.




Supplementary file Size Download File type/resource
GSM1380852_Xe-G-193109-041228.CEL.gz 2.2 Mb (ftp)(http) CEL
Processed data included within Sample table
Processed data are available on Series record

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