|
| Status |
Public on Apr 01, 2008 |
| Title |
unwounded versus wounded drosophila embryos C |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
unwounded drosophila embryos
|
| Organism |
Drosophila melanogaster |
| Characteristics |
unwounded wild-type drosophila embryos
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol followed by RNeasy cleanup
|
| Label |
Cy3
|
| Label protocol |
Amino-allyl reverse transcription labeling protocol
|
| |
|
| Channel 2 |
| Source name |
wounded drosophila embryos
|
| Organism |
Drosophila melanogaster |
| Characteristics |
wounded wild-type drosophila embryos
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol followed by RNeasy cleanup
|
| Label |
Cy5
|
| Label protocol |
Amino-allyl reverse transcription labeling protocol
|
| |
|
| |
| Hybridization protocol |
ARRAY PREHYBRIDIZATION WITH MILK
Updated 09.29.2003
*In 500mL graduated cylinder, measure the following:
370mL Nano-H2O
125mL 20X SSC
5mL 10% SDS
*Weigh out 15grams of powdered non-fat milk and place in beaker (at least 500mL beaker).
*Pour prehybridization solution from beaker into slide dish. Cover with plastic wrap and place in 42C water bath. Water level should be the same level as solution in dish…no higher.
*Allow solution to heat for about 45 minutes.
*Place rack of slides to be prehybridized into preheated solution. Dip up and down a few times to get slides wet. Cover the dish with a metal lid or plastic wrap and let sit (still in the 42C bath) for approximately 1 hour.
*Remove slides from prehybridization solution and immediately place in a dish of Nano-H2O. Dip up and down 15-20 times vigorously.
*Transfer rack of slides to a dish of 100% Isopropanol. Dip up and down 15-20 times vigorously.
*Immediately transfer to centrifuge and spin dry at 500xg for 5 minutes.
HYBRIDIZATION AND WASH PROTOCOL
Updated 09.10.2003
Array (Slide) Examination
*Prescan Array slide with Axon scanner at low resolution.
*Prehybridize Arrays in 3% Milk solution for 1 hour. (See MILK PREHYBRIDIZATION PROTOCOL).
Sample Preparation
*Allow Labeled RNA sample(s) to thaw.
*Add 0.6ul 10% SDS (Do not ice after adding SDS).
*Heat @ 99.9°C for 2 min. on heatblock.
*Centrifuge @ 14,000 RPM for 3 min.
*Cool to RT.
Hybridization Setup
*Place array slide in Hybridization chamber.
*Add 10ul 3X SSC to slide (well away from spotted array).
*Add probe sample onto array area. (Do NOT touch surface with pipette)
*Apply coverslip PROMPTLY over array. (Position carefully and Avoid bubbles)
*Seal hybridization chamber.
*Incubate @ 63°C ~ 16 hrs. by submerging in water bath immediately.
Post-Hybridization Washes
Wash 1A (with SDS): 1X SSC/ 0.03% SDS
*Remove array slide from Hybridization chamber.
*Place slide in Slide rack submerged in wash.
(If coverslip begins to come off, allow it to fall free in wash to avoid scratching array)
*Soak in solution for 2 min.
*Dip slide rack until coverslip(s) falls free.
Wash 1B (NO SDS): 1X SSC
*Dip slide (rack) 15 times to remove excess SDS.
Wash 2: 0.2X SSC
*Soak and Shake @ 60RPM protected from light for 20 min.
Wash 3: 0.05X SSC
*Soak and Shake @ 60RPM protected from light for 10 min.
Note: Immediate transfer of slide(s) through wash steps will avoid drying effects.
Drying
*Centrifuge slide(s) @ 1000RPM for 5 min.
COVERSLIP CLEANING
Updated 10.14.2003
*The following protocol is for cleaning glass coverslips.
*The recipes are for two slide staining dishes with volumes of ~500ml.
*Do not use powdered gloves
*To avoid dust, keep glass slides covered or submerged at all times
*Prepare wash solution:
70g NaOH pellets
420 ml 95% EtOH
280 ml nanopure H20
*Mix solution until NaOH is completely dissolved (add EtOH last).
*Rinse two glass staining dishes thoroughly with DI water. Add racks to staining dish and pour wash solution over coverslips. Cover dishes with dish cover or plastic cling wrap.
*Shake glass coverslips in wash solution for 2 hours using an orbital shaker (~80 rev/min).
*Rinse slides with copious amounts of DI H20 (4 washes/dish) then perform final wash with Nanopure H20.
*Immediately spin down racks with slides in table-top centrifuge w/ 96-well plate carriers at 600 rpm for 5 min.
*Place racks in 50°C vacuum oven for 2 min. under 20 In. Hg vacuum.
*Store in clean, well-labeled boxes to prevent exposure to dust.
|
| Scan protocol |
Contact FHCRC microarray facilities for details
|
| Description |
unwounded versus wounded drosophila embryos C
|
| Data processing |
A Lowess curve was fit to the log-intensity versus log-ratio plot. 20.0% of the data was used to calculate the Lowess fit at each point. This curve was used to adjust the control value for each measurement. If the control channel was lower than 10 then 10 was used instead.
|
| |
|
| Submission date |
Jan 18, 2008 |
| Last update date |
Mar 18, 2008 |
| Contact name |
Mark Owen Winfield |
| E-mail(s) |
Mark.Winfield@bristol.ac.uk
|
| Phone |
44 117 3317086
|
| Organization name |
Bristol University
|
| Department |
Biological Sciences
|
| Street address |
Woodland Road
|
| City |
Bristol |
| ZIP/Postal code |
BS8 1UG |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL1908 |
| Series (2) |
| GSE10207 |
wounded versus nonwounded drosophila embryos |
| GSE10225 |
Expression study of 'hemocyte minus vs hemocyte plus, and wounded vs unwounded hemocyte deficient' drosophila embryos |
|
