2 ug poly-A RNA extracted with Invitrogen fast track
Label
Cy3
Label protocol
standard
Hybridization protocol
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). Add 1 µl COT-1 DNA (8-10 ug/ul) and 1 ul poly A (8-10 ug/ul). 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 20 ul of 2X F-hyb buffer to samples. Load 40 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), for 1 minute in 0.05X SSC, and spin for 3 minutes at 650 rpm to dry. (Refer to NCI Microarray Manual: http://nciarray.nci.nih.gov/reference/nciarrayspacket01-04A.pdf)
Scan protocol
standard
Description
Tumor biopsies from 71 untreated patients with mantle cell lymphoma
Data processing
Values are log2 of 50th Percentile normalized Mean (CY5 int)-Median(CY5 bkg)/Mean (CY3 int)-Median(CY3 bkg). Axon GenePix Pro program was used for Feature extraction.
