129/c57BL/6 male, thy1+ gonocytes isolated from 2 dpp testes and treated with RA for 8hr
Extracted molecule
total RNA
Extraction protocol
Trizol reagent solution
Label
biotin
Label protocol
Approximately 75 ng total RNA from each sample was used to generate cDNA using the Ovation™ RNA Amplification System V2 (NuGEN, San Carlos, CA) according to the manufacturer’s instructions. Briefly, first and second strand cDNA were synthesized, followed by SPIA™ isothermal linear amplification to generate single stranded antisense cDNA. Single stranded antisense cDNA (3.75 ug) was then enzymatically fragmented and end-labeled with biotin using the FL-Ovation™ cDNA Biotin Module V2 (NuGEN, San Carlos, CA) following the manufactures protocol.
Hybridization protocol
To detect changes in expression levels for gene transcripts, we used the Mouse Genome 430v2.0 GeneChip™ array (Affymetrix, Santa Clara, CA). This array contains more than 45,000 probe sets representing 39,000 transcripts and variants, and is currently the most comprehensive GeneChip array available for the mouse. Approximately 3.75 ìg of biotin-labeled, fragmented cDNA was hybridized to each array for 18 h at 45 °C. Washes and post hybridization processing were performed using the Fluidics Station 450 (Affymetrix) according to the manufacturers’ instructions, and the GeneChip™ arrays were scanned using a laser confocal slide scanner (GeneChip Scanner 3000, Affymetrix). Array quality was assessed and images were quantified using GeneChip™ Operating Software (GCOS) v1.2 (Affymetrix).
Scan protocol
To detect changes in expression levels for gene transcripts, we used the Mouse Genome 430v2.0 GeneChip™ array (Affymetrix, Santa Clara, CA). This array contains more than 45,000 probe sets representing 39,000 transcripts and variants, and is currently the most comprehensive GeneChip array available for the mouse. Approximately 3.75 ìg of biotin-labeled, fragmented cDNA was hybridized to each array for 18 h at 45 °C. Washes and post hybridization processing were performed using the Fluidics Station 450 (Affymetrix) according to the manufacturers’ instructions, and the GeneChip™ arrays were scanned using a laser confocal slide scanner (GeneChip Scanner 3000, Affymetrix). Array quality was assessed and images were quantified using GeneChip™ Operating Software (GCOS) v1.2 (Affymetrix).
Description
To detect changes in expression levels for gene transcripts, we used the Mouse Genome 430v2.0 GeneChip™ array (Affymetrix, Santa Clara, CA). This array contains more than 45,000 probe sets representing 39,000 transcripts and variants, and is currently the most comprehensive GeneChip array available for the mouse. Approximately 3.75 ìg of biotin-labeled, fragmented cDNA was hybridized to each array for 18 h at 45 °C. Washes and post hybridization processing were performed using the Fluidics Station 450 (Affymetrix) according to the manufacturers’ instructions, and the GeneChip™ arrays were scanned using a laser confocal slide scanner (GeneChip Scanner 3000, Affymetrix). Array quality was assessed and images were quantified using GeneChip™ Operating Software (GCOS) v1.2 (Affymetrix).
Data processing
For each microarray, probe set signal values were initially normalized by scaling to a target intensity of 125.
