Two weeks after doxycline induction, total RNA was isolated from 10T1/2-tTA cells infected with different combinations of Tbx5, Gata4, and Myocardin lentiviruses using Trizol reagent (Invitrogen) followed by DNase I (Qiagen) treatment. RNA was then recovered by using RNeasy MinElute Cleanup Kit (Qiagen).
Label
biotin
Label protocol
Samples were then processed according to Ambion Message Amp II protocol using the kit and 500ng of total RNA. The Bio-11-UTPs were used for the RNA biotinylation and a final reaction volume of 40uL was used. After labeling and amplification, the samples were then fragmented, hybridized, washed, stained, and scanned using the Affymetrix Genechip™ microarray system. An input amount of 15.0 µg of the labeled cRNA was fragmented and re-checked for concentration. A hybridization cocktail containing Affymetrix spike-in controls and fragmented labeled cRNA was loaded onto the Mouse 430 2.0 GeneChip® array. The array was hybridized for 16 hours at 45°C with a rotation speed of 60 rpm then washed and stained with a strepavidin, R-phycoerythrin conjugate. Signal amplification was measured using the biotinylated antistreptavidin. The stained array was scanned on the Affymetrix GeneChip® Scanner 3000. The images were analyzed and quality control metrics recorded using Affymetrix® GeneChip® Command Console® 3.0 (AGCC).
Hybridization protocol
Samples were then processed according to Ambion Message Amp II protocol using the kit and 500ng of total RNA. The Bio-11-UTPs were used for the RNA biotinylation and a final reaction volume of 40uL was used. After labeling and amplification, the samples were then fragmented, hybridized, washed, stained, and scanned using the Affymetrix Genechip™ microarray system. An input amount of 15.0 µg of the labeled cRNA was fragmented and re-checked for concentration. A hybridization cocktail containing Affymetrix spike-in controls and fragmented labeled cRNA was loaded onto the Mouse 430 2.0 GeneChip® array. The array was hybridized for 16 hours at 45°C with a rotation speed of 60 rpm then washed and stained with a strepavidin, R-phycoerythrin conjugate. Signal amplification was measured using the biotinylated antistreptavidin. The stained array was scanned on the Affymetrix GeneChip® Scanner 3000. The images were analyzed and quality control metrics recorded using Affymetrix® GeneChip® Command Console® 3.0 (AGCC).
Scan protocol
The stained array was scanned on the Affymetrix GeneChip® Scanner 3000. The images were analyzed and quality control metrics recorded using Affymetrix® GeneChip® Command Console® 3.0 (AGCC).
Data processing
Samples were compared by using Affymetrix MG 430 2.0 arrays. Expression data were analyzed by using dChip2008 and GenMAPP software programs. Genes with an absolute fold change =2.0 relative to the LacZ group and genes with a signal intensity of 0 in the LacZ group but not in the transcription factor-treated groups (or vice versa) were subjected to GO analysis. GO terms with P<0.05 and a nested gene change number =4 were considered as statistically significant activation or inhibition. Activated and inhibited gene lists were compared by using the GeneVenn web application.
