Cells were treated with either flagellin or LPS for 1, 2, 4, or 24 hours.
Growth protocol
RAW 264.7 cells were grown in DMEM supplemented with 10% FBS.
Extracted molecule
total RNA
Extraction protocol
Cells were washed twice with PBS and then RNA was harvested according to the TriZol protocol.
Label
Cy5
Label protocol
A total of 2 ug of RNA in a mixture containing 6 ug of random hexamers, 0.01 M dithiothreitol, an aminoallyl-deoxynucleoside triphosphate mixture containing 25 mM each dATP, dCTP, and dGTP, 15 mM dTTP, and 10 mM amini-allyl dUTP (aa-dUTP), reaction buffer, and 400 units of SuperScript III reverse transcriptase at 42C overnight. The RNA template then was hydrolyzed by adding NaOH and EDTA to a final concentration of 0.2 and 0.1 M, respectively, and incubating at 65C for 15 min. Unincorporated aa-UTP was removed with a MinElute column. The probe was eluted with a phosphate elution buffer (4 mM KPO4, pH 8.5, in ultrapure water), dried and resuspended in 0.1 M sodium carbonate buffer (ph 9.0). To couple the amino-allyl cDNA with fluorescent labels, normal human serum-Cy3 or normal human serum-Cy5 was added at room temperature for 1 hour. Uncoupled label was removed using the Qiagen MinElute columns.
Cells were treated with either flagellin or LPS for 1, 2, 4, or 24 hours.
Growth protocol
RAW 264.7 cells were grown in DMEM supplemented with 10% FBS.
Extracted molecule
total RNA
Extraction protocol
Cells were washed twice with PBS and then RNA was harvested according to the TriZol protocol.
Label
Cy3
Label protocol
A total of 2 ug of RNA in a mixture containing 6 ug of random hexamers, 0.01 M dithiothreitol, an aminoallyl-deoxynucleoside triphosphate mixture containing 25 mM each dATP, dCTP, and dGTP, 15 mM dTTP, and 10 mM amini-allyl dUTP (aa-dUTP), reaction buffer, and 400 units of SuperScript III reverse transcriptase at 42C overnight. The RNA template then was hydrolyzed by adding NaOH and EDTA to a final concentration of 0.2 and 0.1 M, respectively, and incubating at 65C for 15 min. Unincorporated aa-UTP was removed with a MinElute column. The probe was eluted with a phosphate elution buffer (4 mM KPO4, pH 8.5, in ultrapure water), dried and resuspended in 0.1 M sodium carbonate buffer (ph 9.0). To couple the amino-allyl cDNA with fluorescent labels, normal human serum-Cy3 or normal human serum-Cy5 was added at room temperature for 1 hour. Uncoupled label was removed using the Qiagen MinElute columns.
Hybridization protocol
Labeled cDNA was hybridized to the Agilent arrays according to the Agilent protocol.
Scan protocol
Hybridizations were scanned on the Axon 4000B scanner with GenePix Pro software.
Data processing
Intensity values were extracted using Feature Extraction 9.5.3.1 from Agilent and then normalized and analyzed using MIDAS and MeV from the TM4 Suite.
