1. Assemble RNA Labeling Reaction: (amounts are per sample to be tested) 1.2µL 10X Iglori Buffer 1.2mL 100% DMSO 1µL .105µM Bio B Primer 1µL .35µM Bio C Primer 2µL Cy-3 (250ng/µL) 1µL 20U/µL T4 RNA Ligase Add 7.4µL Labeling Solution to 5µL of 10µg Total RNA Incubate on ice for 2 hours. 2. After 2 hours of labeling, add 46µL of RNA precipitation solution: (prepare immediately before use) 40µL DEPC Water 5µL 3M Sodium Acetate (pH5.2) 1µL Glycogen Add 150µL of 100% ethanol and finger vortex. Place on ice for 10 minutes. Centrifuge for 10 minutes, max speed @ 4°C. Remove supernatant. Add 150µL of 70% ethanol. Finger vortex. Spin 5 minutes, max speed @ 4°C. Remove ethanol. Briefly spin again at max speed. Remove remaining ethanol with a p20 pipet. Let pellet air dry for 2-3 minutes. Resuspend pellet with 6µL DEPC water.
Hybridization protocol
1. Pipet 35µL of the prehyb solution into one hyb portal of the hyb cap. Prehyb Solution: (prepare immediately before use) Amount appropriate for one array 22.5µL 20X SSC 1.5µL 10% SDS 15µL 10X (2%) BSA 111µL DEPC H2O Make sure there are no bubbles in the hyb chamber. Also take care that none of the prehyb solution comes out of the hyb portal on the other side hyb chamber. Securely tape the hyb portals with Scotch Tape. Place the hyb clamp assembly into a hyb tube fitted with a large kimwipe in such a way that the clamp assembly does not move. Place the hyb tube into a hybridization oven, and allow to rotate for a minimum of 1 hour at 37°C. 2. Prepare hybridization buffer: (amounts are per sample) 17.5µL 2X Hyb Solution (7.5mL 1M Na2HPO4, 150mg BSA, 0.938g Ultrapure SDS, 1.88ml DEPC Water) 4.2µL DI Formamide 7.3µL DEPC Water Add labeled RNA to 29µL of hybridization buffer. 3. Heat sample for 3 minutes at 95°C. 4. Remove prehyb solution from the hyb portals. Add 35µL of sample-hyb mix directly to hyb portals Make sure there are no bubble in the hyb chamber. Also take care that none of the hyb solution comes out of the hyb portal on the other side hyb chamber. 5. Securely tape the hyb portals with Scotch Tape. Place the hyb clamp assembly into a hyb tube fitted with a large kimwipe in such a way that the clamp assembly does not move. Place the hyb tube into a hybridization oven, and allow to rotate overnight at 37°C. 6. Remove hyb solution from the hyb portals. Quickly rinse the chambers with Wash #1 (2X SSC, 0.025% SDS). Remove Wash, and refill chambers with 35µL Wash #1. Incubate at room temp for 3 minutes. 7. Remove Wash #1, and quickly rinse chambers with 35µL Wash #2 (1.6X SSC). Remove Wash, and refill chambers with 35µL Wash #2. Incubate at room temp for 3 minutes. Repeat 2 more times. 8. Remove Wash #2, and quickly rinse chambers with 35µL of ice cold Wash #3 (0.8X SSC) in a cold room. Remove Wash, and refill chambers with 35µL Wash #3. Incubate at 4°C room temp for 3 minutes. Repeat 2 more times. Remove all wash solution. 9. Remove array from hyb clamp, and remove hyb cap. 10. Add 1 or 2 drops of Imaging Solution (from Combimatrix) to array. 11. Carefully clean hyb cover slips with 70% EtOH. 12. Using forceps, place cover slip over semiconductor portion of array, with the white (rough) strips down, making sure no bubbles are trapped. Blot excess solution from edges of cover slip. 13. Keep in the dark until ready to scan.
Scan protocol
Scan with GenePix 4000B microarray scanner.
Description
N/A
Data processing
Hybridization signals extracted using Combimatrix Microarray Imager software. VALUE column represents median-normalized, background-subtracted hybridization signals.
