P493-6 cells were seeded in 6-well plate and treated with doxcycline for 0 or 24 hours.
Growth protocol
P493-6 cells carrying a c-Myc tet-off system were cultured in RPMI medium 1640 containing 10% fetal bovine serum in a humidified incubator with 5% CO2.
Extracted molecule
total RNA
Extraction protocol
Pellet cells by centrifugation. Lyse cells in TRIzol Reagent by repetitive pipetting. Use 1 ml of the reagent per 5-10 × 106 of P493-6 cells. Incubate the homogenized samples for 5 minutes at 15 to 30°C to permit the complete dissociation of nucleoprotein complexes. Add 0.2 ml of chloroform per 1 ml of TRIzol Reagent. Cap sample tubes securely. Shake tubes vigorously by hand for 15 seconds and incubate them at 15 to 30°C for 2 to 3 minutes. Centrifuge the samples at 12,000 × g for 15 minutes at 4°C. Following centrifugation, the mixture separates into a lower red, phenol-chloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase. The volume of the aqueous phase is about 60% of the volume of TRIzol Reagent used for homogenization. Transfer the aqueous phase to a fresh tube. Precipitate the RNA from the aqueous phase by mixing with isopropyl alcohol. Use 0.5 ml of isopropyl alcohol per 1 ml of TRIzol Reagent used for the initial homogenization. Incubate samples at 15 to 30°C for 10 minutes and centrifuge at 12,000 × g for 10 minutes at 4°C. Remove the supernatant. Wash the RNA pellet once with 75% ethanol, adding at least 1 ml of 75% ethanol per 1 ml of TRIzol Reagent used for the initial homogenization. Mix the sample by vortexing and centrifuge at 7,500 × g for 5 minutes at 4°C. At the end of the procedure, air-dry the RNA pellet for 5-10 minutes. Dissolve RNA in 85ul or less RNase-free water by passing the solution a few times through a pipette tip, and incubating for 10 minutes at 55 to 60°C.
Label
Cy3
Label protocol
RNA was amplified and labeled with Cy3 by cDNA Master Mix and Quick Amp Labeling Kit, One-Color (Agilent p/n 5190-0442). And the cRNA was purified by RNeasy Mini Kit (Qiagen p/n 74104).
Hybridization protocol
1. Add 500 μL of nuclease-free water to the vial containing lyophilized 10X Blocking Agent. Mix by gently vortexing. 2. Equilibrate water bath to 60°C. 3. For each microarray, add each of the components as indicated in the tables as below to a 1.5 mL nuclease-free microfuge tube: For 8*60K array Amount, cyanine 3-labeled, linearly amplified cRNA 0.6ug, 10X Blocking Agent 5ul, Nuclease-free water ×ul, 25X Fragmentation Buffer 1ul, Total volume 25ul. 4. Mix well but gently on a vortex mixer. 5. Incubate at 60°C for exactly 30 minutes to fragment RNA. 6. Add 2x GEx Hybridization Buffer HI-RPM to the array to stop the fragmentation reaction. 7. Mix well by careful pipetting. Take care to avoid introducing bubbles. Do not mix on a vortex mixer; mixing on a vortex mixer introduces bubbles. 8. Spin for 1 minute at room temperature at 13,000 rpm in a microcentrifuge to drive the sample off the walls and lid and to aid in bubble reduction. 9. Place sample on ice and load onto the array as soon as possible. 10. Load a clean gasket slide into the Agilent SureHyb chamber base with the label facing up and aligned with the rectangular section of the chamber base. Ensure that the gasket slide is flush with the chamber base and is not ajar. 11. Slowly dispense the volume of hybridization sample onto the gasket well in a “drag and dispense” manner. 12. Slowly place an array “active side” down onto the SureHyb gasket slide, so that the “Agilent”-labeled barcode is facing down and the numeric barcode is facing up. Verify that the sandwich-pair is properly aligned. 13. Place the SureHyb chamber cover onto the sandwiched slides and slide the clamp assembly onto both pieces. 14. Hand-tighten the clamp onto the chamber. 15. Vertically rotate the assembled chamber to wet the gasket and assess the mobility of the bubbles. 16. Place assembled slide chamber in rotisserie in a hybridization oven set to 65°C. Set your hybridization rotator to rotate at 10 rpm. 17. Hybridize at 65°C for 17 hours.
Scan protocol
1. Assemble the slides into an slide holder. 2. Place assembled slide holders into scanner carousel. 3. Verify scan settings for one-color scans. Parameters: Scan region Scan Area (61 x 21.6 mm), Scan resolution (μm) 5, 5μm scanning mode Single Pass, eXtended Dynamic range (selected), Dye channel Green, Green PMT XDR Hi 100%, XDR Lo 10%. 4. Click Scan Slot m-n on the Scan Control main window where the letter m represents the Start slot where the first slide is located and the letter n represents the End slot where the last slide is located.
Description
total RNA (ncRNA and mRNA) Gene expression after 24hr in doxcycline-treated P493-6 cell lines which carry the Myc tet-off system
Data processing
1. Open the Agilent Feature Extraction (FE) software. 2. Add the images (.tif) to be extracted to the FE Project. 3. Set FE Project Properties. 4. Check the Extraction Set Configuration. 5. Save the FE Project (.fep) by selecting File > Save As and browse for desired location. 6. Select Project > Start Extracting and export data to txt. Raw signal intensities were normalized in quantile method by GeneSpring GX v11.5.1. The GeneSpring GX v11.5.1 software package (Agilent Technologies) is used to produce the files LncRNA Expression Profiling Data.xls and mRNA Expression Profiling Data.xls (available on the series record).
