8 protocols
AccessionType
bioassay_data_transformation
The data was analysed using ArrayPro Analyzer - a commercial spot finding and quantification tool produced by Media Cybernetics. The data was normalised using background subtracted normalised protocol.
grow
The Drosophila were maintained on standard cornmeal sucrose agar medium at 25C. The light dark schedule was not controlled for these cultures.
specified_biomaterial_action
Fly cultures were maintained at 25C on standard food. Wandering 3rd instar larvae were collected and put into fresh vials. 3 days after collecting (pupae are then 2-3 days old), the vials were submitted to 3 cycles of heat shock (1 hour at 37C in a water bath, 2 hours at room temperature) and put back at 25C. Heads were dissected for RNA later.
labeling
Direct Labelling of Total RNA

Overview

All materials should be autoclaved and only handled using gloves to avoid RNase contamination. Glassware should be baked at 180 C overnight. MilliQ water and solutions should be treated with DEPC, by adding 1:1000 dilution of DEPC (in fume cupboard), leaving to stand overnight and then autoclaving. The work area can be cleaned using RNase Zap to further limit the risk of RNase contamination. If possible, keep a set of pipettes purely for RNA work. The described protocol is based on the method recommended by BioRobotics (http://www.biorobotics.co.uk/).

Equipment and reagents

dATP, dCTP/dTTP and dGTP, Sigma, cat.no. dNTP-100A
Oligo (dT)15, Promega, cat. no. C1101
DEPC (Diethyl pyrocarbonate), BDH, cat.no. 44170 3D
MilliQ water, DEPC-treated
Cy3 dCTP, Amersham, cat. no. PA 53021
Cy5 dCTP, Amersham, cat. no. PA 55021
RNAsin, Promega, cat. no. 18064-014
First strand buffer, Gibco/BRL, cat. no. 18064-014
Superscript II Reverse Transcriptase, Gibco/BRL, cat. no. 18064-014
0.1M DTT, Gibco/BRL, cat. no. 18064-014
EDTA, BDH, cat. no. 100935V
NaOH, BDH, cat. no. 102525P
Tris-HCl (pH 7.5), BDH, cat. no. 443864E
ArrayHyb Hybridisation Solution, Sigma, cat. no. A7718
Sonicated Salmon Sperm DNA, Amersham, cat. no. 27-4565-01
AutoSeq G-50 column, Amersham, cat. no. 27-5340-01
Micro 20 centrifuge, Hettich
Hot-block, Grant QBT2
Speed Vac, Savant

Procedure

Reverse transcription reaction
Prepare a concentrated stock of low-C dNTP mix:
25 ul of 100 mM dATP
25 ul of 100 mM dGTP
25 ul of 100 mM dTTP
10 ul of 100 mM dCTP
Make to 500 ul with DEPC-treated MilliQ water
Store in small aliquots at -20 C
Mix together 25-50 ug total RNA and DEPC MilliQ water to a total volume of 28 ul in an RNAse-free 1.5 ml tube. Add 1 ul of 500 ng/ml oligo (dT)15 primer.
Incubate at 65 C for 10 minutes in a hot-block to denature RNA tertiary structure, then place on ice.
Mix together the following to make a master mix:
8 ul of 5x first strand buffer
2 ul of conc. low-C dNTP mix
2 ul of 1 mM Cy3 or Cy5 dCTP
2 ul of 0.1 M DTT
0.5 ul of RNAsin
2 ul of Superscript II reverse transcriptase
Add 16.5 ul master mix to each tube of RNA/MilliQ water mixing carefully to avoid bubbles. Do not expose samples to light any more than necessary, ie. wrap in foil when possible.
Incubate at 42 C for 1-2 hours.

Hydrolysis and neutralisation

Stop the reaction by adding 10 ul of 0.5 M EDTA pH8. Hydrolyse the remaining RNA by adding 10 ul of 1 M NaOH and incubating at 65 C for 15 minutes. (Mix equal volumes of 0.5 M EDTA and 1 M NaOH as a stock and then add 20 ul of this mix).
Bring samples to room temperature and add 25 ul of 1 M Tris-HCl (pH 7.5) to neutralise. If required, the labelled probe can be stored at -20 C in the dark at this point.

Probe clean-up

It is important to separate the fluorescently-labelled probe from any unincorporated dye and nucleotides. AutoSeq G-50 columns are quick and easy to use, although Microcon 30 columns (Millipore) or Qiaquick PCR purification columns (Qiagen) work equally well.
Purify probe using an AutoSeq G-50 column as follows:

Reduce volume of probe to approximately 25 ul, by placing in a speed vac with medium heat. With our machine, this takes about 30 mins.
Resuspend the resin in the G-50 column by vortexing gently.
Loosen the cap a quarter turn and snap off the bottom closure.
Place the column in a 1.5 ml tube.
Pre-spin column at 5,000 rpm for 1 min to remove the buffer (see information supplied with the columns for calculating centrifugation speed). Blot the tip of the column dry using a clean paper towel.
Remove the top cap and place column in a new 1.5 ml tube. Pipette the sample onto the centre of the angled surface of the compacted resin bed being careful not to disturb the resin. Do not allow any of the sample to flow around the sides of the bed.
Spin for 1 min at 5,000 rpm. The unincorporated dye and nucleotides should be retained by the column and the purified labelled probe should pass through into the support tube. Discard the column.
Combine the Cy3- and Cy5-labelled probe and blocking agent. I use 2 ul of 10 mg/ml sonicated salmon sperm DNA. Place tube in a speed vac with medium heat and spin until the probe volume is reduced to approximately 5 ul. (Do not evaporate completely, as the sample becomes difficult to resuspend).
Add appropriate volume of hybridisation solution (Sigma ArrayHyb). For use with the Genomic Solutions GeneTAC Hybridisation station, make the probe up to a final volume of 135 ul. Boil for 2 mins to denature probe (hot-block at 100 C). Centrifuge for 1 min at 13,000 rpm and add probe to microarray, avoiding any precipitate.
nucleic_acid_extraction
Small Scale Extraction of Total RNA from Drosophila Samples

Overview:

All materials should be autoclaved and only handled using gloves to avoid RNase contamination. Glassware should be baked at 180C overnight. Water and solutions should be treated with DEPC, by adding 1:1000 dilution of DEPC (in fume cupboard), leaving to stand overnight and then autoclaving. The work area can be cleaned using RNase Zap to further limit the risk of RNase contamination. If possible, it is a good precaution to keep a separate set of pipettes purely for RNA work.
The RNA that is to be labelled must be of high quality. It must be undegraded and contain no genomic DNA contamination. Several extraction methods have been tested for use with Drosophila samples. Extraction using TRIzol gives consistent, reliable results and is considerably cheaper than kit-based products. It is therefore our method of choice.
Poly A+ mRNA constitutes approximately 2% of total RNA from a Drosophila embryo. Labelling of 50 ug total RNA using an oligo(dT) primer gives similar results to approximately 1 ug poly A+ RNA. It is therefore unnecessary to purify poly A+ RNA from the total RNA prep.
The outlined protocol is based on a method from Kevin White's web site (http://quantgen.med.yale.edu/) and is suitable for up to 100 mg of tissue. For microarray experiments performed by the FlyChip facility, we will require 30 ug total RNA per labelling reaction. Please ensure that you have a sufficient amount of tissue before sending us your samples (tissue).
Equipment and reagents

TRIzol, Gibco/BRL, cat. no. 15596-018
DEPC (Diethyl pyrocarbonate), BDH, cat. no. 44170 3D
1.5 ml disposable Polypropylene Pellet Pestle with microtube (Anachem, cat. no. K-749520-0000). Autoclave in DEPC-treated water to ensure that RNase-free
Chloroform, BDH, cat. no. 100775A
Isopropanol, BDH, cat. no. 102246L
DEPC-treated MilliQ water
70% ethanol/DEPC MilliQ water
RNAlater, Ambion, cat. no. 7020
Micro 20 centrifuge, Hettich

Procedure

1. For adult flies, imaginal discs and other tissues, transfer tissue to a 1.5 ml microfuge tube and weigh on microbalance. For embryos, dechorionate first, rinse thoroughly with water and blot off excess before weighing (do not fix!). If samples are ready to be homogenised immediately, skip to step 2. If samples are not yet ready for processing, then either: (i) flash freeze tube in liquid nitrogen then store in -80 C freezer until ready to homogenise. Thaw on ice before continuing with step 2, or (ii) add 5 volumes of RNAlater. The tissue can be stored safely at 25 C for a couple of days, at 4 C for up to a week, and at -20 C or -80 C for at least a month. When ready to continue, remove RNAlater before continuing with step 2.
2. Place sample on ice and add 200 ul TRIzol.
3. Homogenise the sample for 30-60 seconds using a disposable polypropylene pellet pestle and microtube. Avoid making sample hot.
4. Add a further 800 ul TRIzol. At this point the sample can be stored at -80 C until ready to be sent to us on dry ice.
5. Thaw sample on ice. Centrifuge at 13,000 rpm in a microcentriuge for 3 mins at 4 C to pellet debris such as the chorion, vitelline membrane, cuticle etc. Transfer supernatant to a fresh 1.5 ml tube.
6. Add 0.2 volumes chloroform (200 ul), shake vigorously for 15 seconds and incubate at room temperature for 2-3 minutes.
7. Centrifuge at 13,000 rpm for 5 minutes at 4 C.
8. Remove upper phase to a new RNase-free tube, being careful not to touch the interface. Discard tube with lower phase and interface.
9. Add 0.7 volumes of isopropanol to precipitate the RNA. Incubate at room temperature for 5 minutes and then centrifuge at 13,000 rpm for 5 minutes at 4 C.
10. Discard the supernatant and wash the RNA pellet with 1 ml 70% ethanol/DEPC MilliQ water. Centrifuge at 13,000 rpm for 10 minutes at 4 C.
11. Air dry the pellet briefly (leave on work bench). Resuspend in an appropriate volume of DEPC MilliQ water. (I usually use around 100-200 ul DEPC MilliQ water per 100 mg starting tissue). The RNA may dissolve more readily if the DEPC MilliQ water is preheated to 55 C.
12. Verify quality of RNA on an agarose gel and measure the optical density at 260 nm to estimate the RNA concentration and purity. I normally run 1 ul on a 1% agarose gel and dilute 1 ul into 500 ul TE to read the optical density. An OD260/OD280 ratio of 2 indicates that the RNA is pure, but any value between 1.8 and 2.0 is acceptable. RNA concentration can be calculated following the equation:
Concentration (ug/ml) = OD260 x 40 x dilution factor (Value of 40 as 40 ug/ml of RNA has an OD260 reading of 1)
labeling
Direct Labelling of Total RNA

Overview

All materials should be autoclaved and only handled using gloves to avoid RNase contamination. Glassware should be baked at 180 C overnight. MilliQ water and solutions should be treated with DEPC, by adding 1:1000 dilution of DEPC (in fume cupboard), leaving to stand overnight and then autoclaving. The work area can be cleaned using RNase Zap to further limit the risk of RNase contamination. If possible, keep a set of pipettes purely for RNA work. The described protocol is based on the method recommended by BioRobotics (http://www.biorobotics.co.uk/).

Equipment and reagents

dATP, dCTP/dTTP and dGTP, Sigma, cat.no. dNTP-100A
Oligo (dT)15, Promega, cat. no. C1101
DEPC (Diethyl pyrocarbonate), BDH, cat.no. 44170 3D
MilliQ water, DEPC-treated
Cy3 dCTP, Amersham, cat. no. PA 53021
Cy5 dCTP, Amersham, cat. no. PA 55021
RNAsin, Promega, cat. no. 18064-014
First strand buffer, Gibco/BRL, cat. no. 18064-014
Superscript II Reverse Transcriptase, Gibco/BRL, cat. no. 18064-014
0.1M DTT, Gibco/BRL, cat. no. 18064-014
EDTA, BDH, cat. no. 100935V
NaOH, BDH, cat. no. 102525P
Tris-HCl (pH 7.5), BDH, cat. no. 443864E
ArrayHyb Hybridisation Solution, Sigma, cat. no. A7718
Sonicated Salmon Sperm DNA, Amersham, cat. no. 27-4565-01
AutoSeq G-50 column, Amersham, cat. no. 27-5340-01
Micro 20 centrifuge, Hettich
Hot-block, Grant QBT2
Speed Vac, Savant

Procedure

Reverse transcription reaction

Prepare a concentrated stock of low-C dNTP mix:
25 ul of 100 mM dATP
25 ul of 100 mM dGTP
25 ul of 100 mM dTTP
10 ul of 100 mM dCTP
Make to 500 ul with DEPC-treated MilliQ water
Store in small aliquots at -20 C
Mix together 25-50 ug total RNA and DEPC MilliQ water to a total volume of 28 ul in an RNAse-free 1.5 ml tube. Add 1 ul of 500 ng/ml oligo (dT)15 primer.
Incubate at 65 C for 10 minutes in a hot-block to denature RNA tertiary structure, then place on ice.
Mix together the following to make a master mix:
8 ul of 5x first strand buffer
2 ul of conc. low-C dNTP mix
2 ul of 1 mM Cy3 or Cy5 dCTP
2 ul of 0.1 M DTT
0.5 ul of RNAsin
2 ul of Superscript II reverse transcriptase
Add 16.5 ul master mix to each tube of RNA/MilliQ water mixing carefully to avoid bubbles. Do not expose samples to light any more than necessary, ie. wrap in foil when possible.
Incubate at 42 C for 1-2 hours.
Hydrolysis and neutralisation

Stop the reaction by adding 10 ul of 0.5 M EDTA pH8. Hydrolyse the remaining RNA by adding 10 ul of 1 M NaOH and incubating at 65 C for 15 minutes. (Mix equal volumes of 0.5 M EDTA and 1 M NaOH as a stock and then add 20 ul of this mix).
Bring samples to room temperature and add 25 ul of 1 M Tris-HCl (pH 7.5) to neutralise. If required, the labelled probe can be stored at -20 C in the dark at this point.

Probe clean-up
It is important to separate the fluorescently-labelled probe from any unincorporated dye and nucleotides. AutoSeq G-50 columns are quick and easy to use, although Microcon 30 columns (Millipore) or Qiaquick PCR purification columns (Qiagen) work equally well.
Purify probe using an AutoSeq G-50 column as follows:

Reduce volume of probe to approximately 25 ul, by placing in a speed vac with medium heat. With our machine, this takes about 30 mins.
Resuspend the resin in the G-50 column by vortexing gently.
Loosen the cap a quarter turn and snap off the bottom closure.
Place the column in a 1.5 ml tube.
Pre-spin column at 5,000 rpm for 1 min to remove the buffer (see information supplied with the columns for calculating centrifugation speed). Blot the tip of the column dry using a clean paper towel.
Remove the top cap and place column in a new 1.5 ml tube. Pipette the sample onto the centre of the angled surface of the compacted resin bed being careful not to disturb the resin. Do not allow any of the sample to flow around the sides of the bed.
Spin for 1 min at 5,000 rpm. The unincorporated dye and nucleotides should be retained by the column and the purified labelled probe should pass through into the support tube. Discard the column.
Combine the Cy3- and Cy5-labelled probe and blocking agent. I use 2 ul of 10 mg/ml sonicated salmon sperm DNA. Place tube in a speed vac with medium heat and spin until the probe volume is reduced to approximately 5 ul. (Do not evaporate completely, as the sample becomes difficult to resuspend).
Add appropriate volume of hybridisation solution (Sigma ArrayHyb). For use with the Genomic Solutions GeneTAC Hybridisation station, make the probe up to a final volume of 135 ul. Boil for 2 mins to denature probe (hot-block at 100 C). Centrifuge for 1 min at 13,000 rpm and add probe to microarray, avoiding any precipitate.
hybridization
Hybridisation Protocol for Genomic Solutions GeneTAC hybridisation station

Sample preparation

Sonicated salmon sperm DNA directly labelled with Cy3-dCTP or Cy5-dCTP were mixed together. 140 ul of hybridisation buffer was added per slide (ArrayHyb Hybridisation Solution, Sigma, cat. no. A7718). The sample was boiled for 2 mins to denature (hot-block at 100 C), centrifuged for 1 min at 13,000 rpm and added to microarray (see step 2 of hybridisation protocol), avoiding any precipitate.
Solutions

Wash solution 1 in source 1: (1x SSC + 0.03% SDS)
Wash solution 2 in source 2: (0.23x SSC)
Wash solution 3 in source 3: (0.06x SSC)
20x SSC (3 M NaCl, 0.3 M Sodium Citrate, pH 7.0)
Steps

1. O-ring condition: keep at 65 C for 15 minutes, with no agitation
2. Introduce probe: when at a temperature of 65 C
3. Set slide temperature: keep at 65 C for 16 hours, with agitation
4. Wash slides: 5 cycles at 55 C, source 1 flow for 20 seconds, waste 1 hold for 40 seconds
5. Wash slides: 5 cycles at 40 C, source 2 flow for 20 seconds, waste 1 hold for 40 seconds
6. Wash slides: 5 cycles at 25 C, source 3 flow for 20 seconds, waste 1 hold for 40 seconds
image_acquisition
Scanning using the Genomic Solution GeneTac LSIV: 1. Place the slides to be scanned in the carousel. The slide ID should be in the bottom left-hand corner. 2. Start the GeneTac LSIV software by double clicking on the GeneTac icon. The software will open and you can now start scanning. 3. Perform a preview scan to obtain a low resolution image, then adjust the scan area such that the entire microarray is included. 4. Using the preview scan option again, adjust the Cy3 and Cy5 'Gain' settings so that the Cy3 and Cy5 channels are of equal intensity. 5. Once the scan settings have been identified you can then perform a real scan. The images will be saved automatically in the location of your choosing.