Cells were grown at 25 °C in Shields and Sang M3 medium (Sigma) supplemented with tissue culture-grade yeast extract (1 g/l), bactopeptone (2.5 g/l), human insulin (5 µg/ml final concentration), and fly extract (DA Currie, MJ Milner, and CW Evans, The growth and differentiation in vitro of leg and wing imaginal disc cells from Drosophila melanogaster. Development 1988 102: 805-814) (2.5% final concentration), and fetal calf serum (heat-inactivated 1 hr at 56º, 10% final concentration).
Extracted molecule
total RNA
Extraction protocol
Pellet cells by centrifugation in 50 ml Corning tube. Resuspend/wash in 5 ml PBS (put into 15 ml Corning tube). Pellet cells again by centrifugation. Remove supernatant. Lyse cells in TRIzol reagent (.75ml per 5-10 E7 cells) by repetitive pipetting. Incubate at room temp. for 5 minutes. Add .2 ml of chloroform per .75 ml of TRIzol used initially. Shake tubes vigorously for 15 seconds Incubate at room temp. for 2 minutes. Divide into 2 eppendorf tubes per sample(~about 1.267 ml per tube). Centrifuge for 15 minutes at 4 °C at 10,000 x g. Transfer the top aqueous phases to clean tubes. Precipitate the RNA from the aqueous phase by adding .5 ml of isopropenal per .75 ml of TRIzol used initially. Invert tubes once. Incubate samples at room temp. for 10 minutes. Centrifuge for 10 minutes at 4 °C at 10,000 x g. Remove supernatant. Wash RNA pellet once with 75% ethanol, 1 ml per .75 ml of TRIzol used initially. Vortex. Centrifuge at 7,500 x g for 5 minutes at 4 °C. Let pellet air dry for 10 minutes. Dissolve in RNase-free water (DEP treated). Incubate at 30 °C overnight .
Label
Alexa Fluor 647
Label protocol
REVERSE TRANSCRIPTION 1. Use 20 ug of total RNA per reaction with Array50 dendrimer kit. 2. Mix the following reagents in an RNase-free microcentrifuge tube: 20 ug RNA, 1 ul RT primer (for Alexa Fluor 647 dendrimer), nuclease-free water to 6 ul total volume. 3. Mix briefly, and centrifuge briefly. 4. Incubate for 5 minutes at 80 °C. 5. Incubate on ice for 3-5 minutes immediately. 6. Centrifuge briefly, and return to ice. 7. Prepare a reaction mix in a microcentrifuge tube on ice: 2 ul 5X RT buffer, 1 ul 0.1 M DTT, 0.5 ul Superase-in RNase inhibitor (kit), 0.5 ul dNTP mix (kit), 0.5 ul Invitrogen SuperScriptTM III RNase H- Reverse Transcriptase (100 units) (total volume 4.5 ul). 8. Mix gently (do not vortex), and centrifuge briefly. 9. Add 4.5 ul reaction mix to 6 ul RNA-primer annealing reaction (from steps 1-6 above). Mix gently. 10. Incubate for 3 hours at 46 °C. RNA HYDROLYSIS 1. Add 1 ul 1 M NaOH/100 mM EDTA. 2. Incubate at 65 °C for 10 minutes to stop the reaction and degrade the RNA. 3. Add 1.2 ul 2 M Tris, pH 7.5 to neutralize the reaction. NOTE: Label is introduced during the hybridization; see hybridization protocol below.
Cells were grown at 25 °C in Shields and Sang M3 medium (Sigma) supplemented with tissue culture-grade yeast extract (1 g/l), bactopeptone (2.5 g/l), human insulin (5 µg/ml final concentration), and fly extract (DA Currie, MJ Milner, and CW Evans, The growth and differentiation in vitro of leg and wing imaginal disc cells from Drosophila melanogaster. Development 1988 102: 805-814) (2.5% final concentration), and fetal calf serum (heat-inactivated 1 hr at 56º, 10% final concentration).
Extracted molecule
total RNA
Extraction protocol
Pellet cells by centrifugation in 50 ml Corning tube. Resuspend/wash in 5 ml PBS (put into 15 ml Corning tube). Pellet cells again by centrifugation. Remove supernatant. Lyse cells in TRIzol reagent (.75ml per 5-10 E7 cells) by repetitive pipetting. Incubate at room temp. for 5 minutes. Add .2 ml of chloroform per .75 ml of TRIzol used initially. Shake tubes vigorously for 15 seconds Incubate at room temp. for 2 minutes. Divide into 2 eppendorf tubes per sample(~about 1.267 ml per tube). Centrifuge for 15 minutes at 4 °C at 10,000 x g. Transfer the top aqueous phases to clean tubes. Precipitate the RNA from the aqueous phase by adding .5 ml of isopropenal per .75 ml of TRIzol used initially. Invert tubes once. Incubate samples at room temp. for 10 minutes. Centrifuge for 10 minutes at 4 °C at 10,000 x g. Remove supernatant. Wash RNA pellet once with 75% ethanol, 1 ml per .75 ml of TRIzol used initially. Vortex. Centrifuge at 7,500 x g for 5 minutes at 4 °C. Let pellet air dry for 10 minutes. Dissolve in RNase-free water (DEP treated). Incubate at 30 °C overnight .
Label
Alexa Fluor 546
Label protocol
REVERSE TRANSCRIPTION 1. Use 20 ug of total RNA per reaction with Array50 dendrimer kit. 2. Mix the following reagents in an RNase-free microcentrifuge tube: 20 ug RNA, 1 ul RT primer (for Alexa Fluor 546 dendrimer), nuclease-free water to 6 ul total volume. 3. Mix briefly, and centrifuge briefly. 4. Incubate for 5 minutes at 80 °C. 5. Incubate on ice for 3-5 minutes immediately. 6. Centrifuge briefly, and return to ice. 7. Prepare a reaction mix in a microcentrifuge tube on ice: 2 ul 5X RT buffer, 1 ul 0.1 M DTT, 0.5 ul Superase-in RNase inhibitor (kit), 0.5 ul dNTP mix (kit), 0.5 ul Invitrogen SuperScriptTM III RNase H- Reverse Transcriptase (100 units) (total volume 4.5 ul). 8. Mix gently (do not vortex), and centrifuge briefly. 9. Add 4.5 ul reaction mix to 6 ul RNA-primer annealing reaction (from steps 1-6 above). Mix gently. 10. Incubate for 3 hours at 46 °C. RNA HYDROLYSIS 1. Add 1 ul 1 M NaOH/100 mM EDTA. 2. Incubate at 65 °C for 10 minutes to stop the reaction and degrade the RNA. 3. Add 1.2 ul 2 M Tris, pH 7.5 to neutralize the reaction. NOTE: Label is introduced during the hybridization; see hybridization protocol below.
Hybridization protocol
PRE-HYBRIDIZATION. 1. Prepare 50 ml pre-hybridization buffer (5x SSC/0.1% SDS/1% I-block) in a 50 ml screw cap centrifuge tube, and bring to 55 °C. 2. Incubate microarray slide in pre-hybridization buffer at 55 °C for 1.5 hours. 3. Transfer microarray slide to 50 ml tube filled with water and invert gently for 1 minute. Then transfer to new tube with fresh water to wash two times. 4. Centrifuge at 500 rcf for 4 minutes to dry (label end down). cDNA HYBRIDIZATION. 1. Prepare the following cDNA hybridization mix: 12.7 ul of each cDNA synthesis reaction mix, 2.1 ul nuclease-free water, 2.7 ul 2x Formamide-Based hybridization buffer (kit) (total volume 55 ul). 2. Mix (by gentle vortexing) and briefly spin the hybridization mix. 3. Incubate at 80 °C for 10 minutes. 4. Incubate at 55 °C until it is loaded on the microarray slide. 5. Pre-warm the microarray on a slide warmer. 6. Assemble the hybridization solution (55 ul) to the center of the slide. Carefully lower a LifterSlip onto the microarray avoiding bubbles. Place the slide face-up in a hybridization chamber. Pipette 20 μl of water into the wells at either end of the hybridization chamber. Seal the chamber. 7. Submerge the hybridization chamber in a 43 °C water bath and incubate for 14-17 hours. POST-cDNA HYBRIDIZATION WASHES. 1. Disassemble the hybridization chamber. 2. Remove cover slip by immersing the slide in a pre-warmed beaker filled with 400 ml 2x SSC/0.2% SDS at 42-43 °C. 3. Incubate slide in a 50 ml tube of pre-warmed 2x SSC/0.2% SDS at 42 °C for 10-15 minutes. 4. Wash in 2x SSC at room temperature for 10-15 minutes. 5. Wash in 0.2x SSC room temperature for 10-15 minutes. 6. Centrifuge at 500 rcf for 4 minutes to dry. DENDRIMER HYBRIDIZATION. 1. Thaw the 3DNA Capture Reagents (dendrimer solutions) that correspond to the primers used for cDNA synthesis in the dark for 20 minutes. Vortex, spin briefly, and incubate at 55 °C in the dark for 10 minutes. 2. At the same time, thaw 2X Formamide-based hybridization buffer by incubating it at 43 °C. 3. Prepare dendrimer hybridization solution by mixing the following kit components in a microcentrifuge tube: 2.5 ul Alexa Fluor 546 3DNA Array Capture Reagent, 2.5 ul Alexa Fluor 647 3DNA Array Capture Reagent, 27.5 ul 2x formamide-based hybridization buffer, 22.5 ul nuclease-free water (total volume 55 ul). 4. Vortex gently and centrifuge briefly. 5. Incubate at 80 °C for 10 minutes. 6. Assemble the hybridization as for the first hybridization. 7. Submerge the hybridization chamber in a 43 °C water bath and incubate for 5 hours. POST-DENDRIMER HYBRIDIZATION WASHES. 1. Disassemble the hybridization chamber. 2. Remove cover slip by immersing the slide in a pre-warmed beaker filled with 400 ml 2x SSC/0.2% SDS at 65 °C. 3. Incubate slide in a 50 ml tube of pre-warmed 2x SSC/0.2% SDS at 65 °C for 10-15 minutes. 4. Wash in 2x SSC at room temperature for 10-15 minutes. 5. Wash in 0.2x SSC room temperature for 10-15 minutes. 6. Centrifuge at 500 rcf for 4 minutes to dry. 7. Scan as soon as possible.
Scan protocol
The hybridized slides were scanned immediately after hybridization with Genepix Scanner 4000. The scan was done with PMT 650 for both channels.
Description
The experiment compares transcripts from control and ecdysone-treated cells, using a dendrimer hybridization procedure.
Data processing
Raw data was extracted from the scanned image using GenePix 6.0 software, and then normalized using OLIN with 'subtract' background correction (http://itb.biologie.hu-berlin.de/~futschik/software/R/OLIN/). Features flagged as "bad" manually or by GenePix were assigned a value of "null" in the VALUE, CH1_NORM, and CH2_NORM columns.
