Tissue: blood Cell type: monocytes Disease state: Normal
Sample Extraction Protocol Other: Total RNA was prepared using RNAeasy kit (Qiagen, Valencia, CA).
Invitrogen cDNA Labeling Kit - cy5 Other: Labeled sample with Cy5 using a NHS-ester coupling reaction following the manufacturer's cDNA Labeling Kit protocol (Invitrogen).
Hybridization Protocol Other: For pre-hybrization, apply 40 ul of pre-hybridization buffer (5X SSC, 0.1% SDS, 1% BSA) to the array and incubate at 42°C for at least 30 minutes and up to an hour. Wash off the pre-hybridization solution by rapidly plunging the slide in distilled water for 2 minutes, then transfer slide to 100% isopropanol for 2 minutes. Allow slide to air dry completely prior to use. (Can spin dry if in a rush.) (NOTE: Do not exceed 1 hour after pre-hybridization/drying before setting up hybridization.) For hybridization, combine Cy3 and Cy5 labeled targets together (~9 ul recovered for each). Denature target at 100°C for 1 minute, then snap cool on ice. (Final volume should be about 20 ul.) Make fresh 2X Formamide hybridization buffer (50% formamide, 10X SSC, 0.2% SDS) and warm to 42°C just before adding to samples. Add 40 ul of 2X F-hyb buffer and 20ul water to samples. Load 80 ul sample onto microarray. Add 20 ul of 3X SSC to wells in hyb chamber to maintain humidity. Incubate overnight (12-16 hours) at 42°C in water bath or hybridization oven. After hybridization of slides, wash slides for 2 minute in 2X SSC with 0.1% SDS (with occasional plunging), for 2 minute in 1X SSC (with occasional plunging), for 2 minutes in 0.2X SSC (with occasional plunging), and spin for 3 minutes at 650 rpm to dry.
mAdb Data Processing Protocol (v. 2) Calculation Method: After background correction, ratios were median centered based on signals greater than 100 in both channels and linear transformed to obtain the log and linear values given in the data table.