disease state: Control tissue: peripheral blood age (yr): 24 gender: female
Extracted molecule
total RNA
Extraction protocol
For human samples, blood was drawn into a PaxGene tube and RNA extracted per manufacterer's protocol. For cell line, RNA was extracted using Trizol per manufacturer's instruction.
Label
Cy5
Label protocol
5 µg of total RNA were primed with 6 µl of Oligo dT at 70°C for 10 min, then reversed transcribed at 42°C for 2 h in the presence of 400 U SuperScript III RTase (Invitrogen), and 200um dATP, 200um dGTP, 200um dCTP, 51um dTTP and 149um AAdUTP. Samples were treated with RNAse H and cleaned with Qiagen PCR-cleanup kit. Samples were coupled to Cy3 or Cy5 dye (Amersham) in a 0.1M sodium bicarbonate buffer, incubated for 1h.
For human samples, blood was drawn into a PaxGene tube and RNA extracted per manufacterer's protocol. For cell line, RNA was extracted using Trizol per manufacturer's instruction.
Label
Cy3
Label protocol
5 µg of total RNA were primed with 6 µl of Oligo dT at 70°C for 10 min, then reversed transcribed at 42°C for 2 h in the presence of 400 U SuperScript III RTase (Invitrogen), and 200um dATP, 200um dGTP, 200um dCTP, 51um dTTP and 149um AAdUTP. Samples were treated with RNAse H and cleaned with Qiagen PCR-cleanup kit. Samples were coupled to Cy3 or Cy5 dye (Amersham) in a 0.1M sodium bicarbonate buffer, incubated for 1h.
Hybridization protocol
Arrays were treated with 0.2% SDS and soaked in prehybridization solution (1% BSA, 5XSSC and 0.1%SDS) for 45 minutes at 55°C. Dye-labeled samples were mixed 1:1 with hybridization solution (50% formamide, 10X SSC, 0.2% SDS) and 1ul polyA RNA. Each sample was heatshocked at 100°C for 2 minutes, loaded onto the array and hybridized at 42°C for 16 hours. Arrays were washed in descending concentrations of 1: 2X SSC, 0.1% SDS; 2: 1X SSC; 3: 0.1x SSC.
Scan protocol
Arrays were scanned into the GenePix Pro4.1 Software using a 400B scanner (Axon Instruments, Union City, CA).
Description
control cohort sample 02
Data processing
Data presented here are single channel medians. Intensity values were normalized to whole array intensity sum=10,000
