Qiagen RNeasy Kit 1. Disrupt and homogenize the sample immediately using Buffer RLT (β-ME added) and a Polytron for 40 sec. Starting material Volume of Buffer RLT Up to 20 mg 350 μL 20 to 30 mg or tissue is difficult to lyse 600 μL 2. Centrifuge lysate for 3 min at maximum speed in a microcentrifuge, and transfer the supernatant into a new Eppendorf tube. 3. Add 1 volume (350 or 600 μL) of 70% ethanol to the cleared lysate, and mix well by pipetting. 4. Apply 700 μL of the sample to an RNeasy mini spin column sitting in a 2-mL collection tube. 5. Centrifuge for 15 sec at maximum speed. Discard flow-through, and reuse the collection tube. 6. Apply aliquots successively onto the RNeasy column and centrifuge as above. Discard flow-through and reuse the collection tube. 7. Pipet 700 μL Buffer RW1 onto the RNeasy column, incubate RNeasy column for 5 min, and centrifuge for 15 sec at maximum speed. Discard flow-through and the collection tube. 8. Transfer RNeasy column to a new 2-mL collection tube. 9. Pipet 500 μL Buffer RPE onto RNeasy column, and centrifuge for 15 sec at maximum speed to wash. Discard flow-through and reuse the collection tube. 10. Pipet 500 μL Buffer RPE onto RNeasy column, and centrifuge for 2 min at maximum speed to dry the RNeasy membrane. Discard flow-through and the collection tube. Note: Residual ethanol may interfere with subsequent reaction. Remove the RNeasy column so that the column does not contact the flow-through as this will result in carryover of ethanol. 11. (Optional): Place the RNeasy spin column in a new 2-mL collection tube, and discard the old collection tube with filtrate. Centrifuge at full speed for 1 min. 12. Transfer RNeasy column into a new 1.5-mL Eppendorf tube (remove the lid) and pipet 30 μL of DEPC water directly onto the RNeasy membrane. Centrifuge for 1 min at maximum speed to elute. 13. Pipet 30 μL of DEPC water successively directly onto the RNeasy membrane. Centrifuge for 5 min at maximum speed to elute. 14. Transfer elute to a new Eppendorf tube.
Label
Biotin
Label protocol
Reverse Transcription to Synthesize First Strand cDNA: 1. Bring RNA samples to 11 µl with RNase-free water (50-500 ng) 2. Add 9 µl of RT Master Mix (mix well and centrifuge briefly): T7 oligo(dT) primer (1 µl), 10X first strand buffer (2 µl), dNTP mix (4 µl), RNase inhibitor (1 µl), ArrayScript (1 µl), 3. Incubate at 42°C for 2 hrs (use a thermal cycler with the lid off or heated to the same temperature) 4. Place tubes on ice and immediately proceed to the next step Second Strand cDNA Synthesis: 1. Add 80 µl of Second Strand Master Mix to each sample (vortex and spin down): Nuclease-free water (63 µl), 10X Second Strand Buffer (10 µl), dNTP mix (4 µl), DNA polymerase (2 µl), RNase H (1 µl) 2. Place at 16°C for 2 hrs (make sure the block is pre-cooled and the lid is off or at the same temperature) 3. Pre-heat nuclease-free water to 50-55°C for the next step 4. Place tubes on ice and proceed to the next step (the reactions can be frozen at -20°C overnight, but it is better to purify them first) cDNA Purification: 1. Pre-heat nuclease-free water to 50-55°C for at least 10 min 2. Check the cDNA Binding Buffer for precipitation – place at 37°C for up to 10 min and vortex vigorously; cool to RT before use 3. Add 250 µl of cDNA Binding Buffer to each sample, mix by pipetting up and down and quickly spin; proceed quickly to the next step 4. Check that the filter is firmly seated in its tube 5. Pipet the cDNA sample with the cDNA Binding Buffer onto the center of the cDNA Filter Cartridge 6. Centrifuge at 10,000 rpm for 1 min 7. Discard flow-through and replace the cDNA Filter cartridge in the wash tube 8. Make sure 100% EtOH has been added to the Wash Buffer 9. Apply 500 µl of Wash Buffer to each sample 10. Centrifuge at 10,000 rpm for 1 min; discard flow-through 11. Centrifuge at 10,000 rpm for 1 min 12. Transfer the cDNA Filter Cartridge to a cRNA Elution Tube 13. Apply 10 µl of pre-heated nuclease-free water to the cDNA Filter Cartridge 14. Incubate at RT for 2 min 15. Centrifuge at 10,000 rpm for 1.5 min 16. Apply 9 µl of pre-heated nuclease-free water to the cDNA Filter Cartridge 17. Incubate at RT for 2 min 18. Centrifuge at 10,000 rpm for 2 min 19. The eluate is now in about 17.5 µl and it can be stored at -20°C if needed (better to proceed to the next step immediately) In vitro Transcription to Synthesize cRNA: 1. Prepare the IVT Master Mix at room temperature: T7 10x Reaction Buffer (2.5 µl), T7 Enzyme Mix (2.5 µl), Biotin-NTP Mix (2.5 µl) 2. Transfer 17.5 µl of sample to a new 0.2 ml tube 3. Add 7.5 µl of the IVT Master Mix to each sample 4. Place tubes at 37°C for 14 hrs in a thermal cycler (add a 4°C hold at the end) Day 2: 5. Add 75 µl of nuclease-free water to each sample 6. Proceed to the RNA purification step cRNA Purification: 1. Pre-heat nuclease-free water to 50-55°C 2. Add 350 µl of cRNA binding buffer to each sample; proceed immediately to next step 3. Add 250 µl of 100% EtOH to each cRNA sample; mix by pipetting up and down; proceed immediately to the next step 4. Pipet the mixture from above onto the cRNA Filter Cartridge (one per sample) 5. Centrifuge at 10,000 rpm for 1 min; discard flow-through 6. Add 650 µl of Wash Buffer to each filter 7. Centrifuge at 10,000 rpm for 1 min; discard flow-through 8. Centrifuge at 10,000 rpm for 1 min to dry 9. Transfer the Filter cartridge to a new collection tube 10. Add 100 µl of pre-heated nuclease-free water to the center of the filter 11. Incubate at RT for 2 min 12. Centrifuge at 10,000 rpm for 1.5 min
Hybridization protocol
Preheat the oven (with rocking platform) to 58°C. Mix with Hyb Reagents 1. Prepare cRNA samples (dried down, if necessary to achieve required concentration). 2. To 1.5 μg RNA, add RNase-free water up to 10 μL and mix. 3. Leave at RT for 10 minutes to resuspend cRNA. 4. Place the GEX-HYB and GEX-HCB tubes in the 58°C oven for 10 minutes to dissolve any salts that may have precipitated in storage. Inspect the solution; if any salts remain undissolved, incubate at 58°C for another 10 minutes. After allowing to cool to room temperature, mix thoroughly before using. 5. Add 20 μL GEX-HYB to each cRNA sample. Set Up Hybridization 1. Place Illumina Hyb Chamber Gaskets into BeadChip Hyb Chamber. 2. Dispense 200 μL GEX-HCB into each of the two humidifying buffer reservoirs in each Hyb Chamber. 3. Only add buffer to chambers that will be used. 4. Seal Hyb Chamber with lid and keep on bench at room temperature (~22°C) until ready to load BeadChips into Hyb Chamber. 5. Remove all BeadChips from their packages. 6. Holding BeadChip by coverseal tab with tweezers using powder-free gloved hands, slide BeadChip into Hyb Chamber Insert such that the barcode lines up with barcode symbol on the Insert. 7. Preheat the assay sample at 65°C for 5 minutes. 8. Briefly vortex, then briefly centrifuge to collect the liquid in the bottom of the tube. Allow sample to cool to room temperature before using. Pipet sample immediately after cooling to room temp. 9. Load Hyb Chamber Inserts containing BeadChips into the Hyb Chamber with barcode on BeadChip aligned to barcode symbol on the Hyb Chamber. 10. Dispense 30 μL assay sample onto the large sample port of each array. 11. Seal lid onto the Hyb Chamber carefully to avoid dislodging the Hyb Chamber Insert(s). 12. Incubate for 16-20 hours at 58°C with rocker speed at 5. ] Caution: Proceed to “Prepare for High-Temp Wash & Overnight Incubation” on page 2 before stopping for the day. 13. Prepare for High-Temp Wash & Overnight Incubation 14. Prepare 1X High-Temp Wash buffer (add 50 mL 10X stock to 450 mL RNAse-free water). 15. Place waterbath insert into heat block, and add 500 mL prepared 1X High-Temp Wash buffer. 16. Set heat block temp to 55°C and pre-warm High-Temp Wash buffer to that temperature. 17. Close heat block lid and leave overnight. Prepare Reagents 1. The next day, make Wash E1BC solution (add 3 mL E1BC buffer to 1 L RNase-free water). 2. Pre-warm Block E1 buffer (4 mL/chip) to room temperature. 3. Prepare Block E1 buffer (2 mL/chip) with streptavidin-Cy3 (2 μL of 1 mg/mL stock per chip). Use a single conical tube for all BeadChips. Store in dark until detection step. Room-Temp Incubation 1. Remove Hyb Chamber from oven and disassemble. Caution: When removing the coverseal, some splattering may occur. To avoid contact with the buffer, which contains formamide, remove the seals in a shielded area such as a fume hood with the sash lowered. 2. Remove coverseal from the BeadChip. Using tweezers or powder-free gloved hands, place the BeadChip into the slide rack submerged in the staining dish containing 250 mL Wash E1BC solution. 3. Repeat disassembly and placement in E1BC solution for all BeadChips. 4. Using the slide rack handle, transfer the rack into the Hybex Waterbath insert containing High-Temp Wash buffer. High-Temp Wash 1. Incubate static for 10 minutes with the Hybex lid closed. 2. During the 10-minute High-Temp Wash buffer incubation, add fresh 250 mL Wash E1BC solution to a clean staining dish. 3. After the 10-minute High-Temp Wash buffer incubation is complete, immediately transfer the slide rack into the staining dish containing E1BC. 4. Briefly agitate using rack, then shake on orbital shaker for 5 minutes at the highest speed possible without allowing solution to splash out of dish. Ethanol Wash 1. Transfer rack to a clean staining dish containing 250 mL 100% Ethanol (use fresh from Ethanol source bottle). 2. Briefly agitate using rack handle, then shake on orbital shaker for 10 minutes. 2nd Room-Temp Wash 1. Transfer rack to a clean staining dish containing fresh 250 mL Wash E1BC solution. 2. Briefly agitate using rack handle, then shake on orbital shaker for 2 minutes. Block 1. Pipette 4 mL Block E1 buffer into the Wash Tray(s). 2. Transfer the BeadChip, face up into BeadChip Wash Tray(s) on rocker. 3. Rock at medium speed for 10 minutes. Detect 1. Pipette 2 mL Block E1 buffer + streptavidin-Cy3 into fresh Wash Tray(s). 2. Transfer the BeadChip, face up into Wash Tray(s) on rocker. 3. Place cover on tray and rock at medium speed for 10 minutes. 3rd Room-Temp Wash 1. Add 250 mL of Wash E1BC solution to a clean staining dish. 2. Transfer the BeadChip to the slide rack submerged in the staining dish. 3. Briefly agitate using rack, and then shake at RT on orbital shaker for 5 minutes. Dry 1. Prepare centrifuge with plateholders, paper towels and balance rack. Set speed to 275 rcf. 2. Transfer rack of BeadChips from staining dish to centrifuge and spin at RT for 4 minutes. 3. Store dry chips in slide box until scanned.
Scan protocol
Beadchips were scanned at the same time on an Illumina 500GX Beadstation using Illumina BeadScan v. 3 software
Description
Medulloblastoma
Data processing
Data was interpreted in GenomeStudio v. 1.0.2 and quantile normalized
