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Sample GSM178086 Query DataSets for GSM178086
Status Public on Oct 31, 2007
Title Newborn Mouse Ovary_LHX8 Null_3
Sample type RNA
 
Source name Postnatal Day 0.5 Ovarian Tissue LHX8 Null
Organism Mus musculus
Characteristics Background Srain: C57/BL6/129SVEV Hybrid
Gender: Female
Tissue: Ovary
Age: .5 Days Postnatal
Biomaterial provider Baylor College of Medicine- Aleksandar Rajkovic Laboratory
Treatment protocol Ovaries were dissected from day 0.5 animals and placed in RNAlater (Ambion) for 24 hours at 4 degrees Celsius. After 24 hours, the RNAlater was removed and the ovaries were frozen on dry ice and placed in a -80 degree freezer.
Extracted molecule total RNA
Extraction protocol 50 ovaries were pooled per affymetrix array. RNA was isolated using Qiagen RNeasy mini kits. On column DNase digestion was used. RNA was eluted in RNase free water and frozen at -80 degrees Celsius until sample quality could be assessed with the Agilent Bioanalyzer system
Label Biotin
Label protocol Test RNA sample on NanoDrop. Concentration must be 500_g/µL or more. Calculate the volume of RNA sample necessary to start protocol with 5µg. i.e. (5 µg) / (NanoDrop concentration reading in µg/µL) = µL

RNA x uL
DEPC H20 10-x uL
oligo T7 2.0 µL

1. Add all components together in a 0.2µL strip tube.
2. Mix well and spin down.
3. Place in thermalcycler at 70°C for 10 minutes. Should be warming up to 70°C while you prep. the samples.
4. Allow thermalcycler to ramp down to 4°C.
5. 4°C for 2 minutes. Spin down and put on ice.
6. Prepare the 1st Strand MasterMix.

Reverse Transcription (RT) 1st Strand

You should have thawed and vortexed the components during the 70°C-10 minute step.
5X 1st Strand Mix (4.0 µL) /sample
0.1M DTT (2.0 µL) /sample
10mM dNTPs (1.0 µL) /sample
7.0 µL total
1. Add 7.0 µL 1st Strand MasterMix to each sample. Mix.
2. Put on thermalcycler 42°C-1 Hour. Set timer for 2 minutes.
(This setting is actually programmed for 42°C for 1 hour and 3 minutes, to compensate for the extra 2
minutes at the beginning before adding the SuperScript II.
3. After 2 minutes at 42°C add 1.0 µL of SSII enzyme. (Addition should be done on thermalcycler)
4. The rest of the program will run, 1 hour at 42°C.
5. Proceed to 2nd Strand.
(components should be thawed ~10 minutes before thermalcycler finishes.)

Reverse Transcription (RT) 2nd Strand

DEPC H20 91.0 µL /sample
5X 2nd Strand Mix 30.0 µL /sample
E. coli DNA Polymerase I (DNA Pol I) 4.0 µL /sample
10mM dNTPs 3.0 µL /sample
E. coli DNA ligase (ligase) 1.0 µL /sample
RNase H 1.0 µL /sample

1. Put samples on thermalcycler, 16°C for 2 hours.
2. At 2 hours, add 2.0 µL of T4 DNA Polymerase. Mix.
3. Run additional 5 minutes at 16°C.
4. Transfer samples to 1.5mL tube with 10.0 µL of 0.5M EDTA. Vortex.
5. Use cDNA Clean-Up Module to clean up samples.

Clean-Up of Double Stranded cDNA

Necessary components:
1.5mL tube with cDNA and EDTA, mixed.
cDNA Binding Buffer
cDNA Wash Buffer (ethanol added by MCF)
cDNA Elution Buffer
cDNA clean-up column (in 4°C Deli Fridge)
Empty Collection Tube (provided in clean-up module)
Empty 1.5mL tube labeled with sample name for collecting final eluate.
MicroFuge 18 Centrifuge to spin down samples.

cDNA Wash Buffer is supplied as a concentrate and you have to add ethanol before using. Check the tube before use to make sure that etOH has been added.

1. Label Collection Tube with sample name (on side).
2. Add 600.0 µL of cDNA Binding Buffer (yellow) into tube with sample and EDTA.
3. Vortex. Spin down briefly (2 sec.)
4. Add 500.0 µL sample-Binding Buffer mix onto column.
5. Spin in MicroFuge 18 Centrifuge, 14,000rpm for 1 minute.
6. Pour eluate down sink. Keep same collection tube.
7. Add the remaining cDNA-Binding Buffer mix onto column.
8. Spin 14K rpm for 1 minute.
9. Discard that collection tube and place column into new, labeled tube.
10. Add 720.0 µL cDNA Wash Buffer (ETOH added) to the column.
11. Spin 14K rpm for 1 minute.
12. Discard eluate into sink. Replace column into collection tube.
13. Spin empty column with the lid open for 5 minutes at 14K rpm.
14. Place column into labeled 1.5mL tube. Discard collection tube.
15. Air dry column for an additional minute (cover with KimWipe).
16. Add 14.0 µL Elution Buffer onto the center of the column.
17. Let sit at room temp for 1 minute.
18. Spin at 14K for 1 minute.
19. Discard column.
20. Measure the eluate volume.
21. Bring volume up to 20.0 µL with DEPC H20.
22. Store at -20ºC until ready to proceed with IVT step.

InVitro Transcription Reaction (IVT): production of biotin-labeled cRNA

cDNA should be at 20.0 µL before adding IVT MasterMix components.
IVT kit components should be thawed to room temp. ~20 minutes before using.
10X IVT Labeling Buffer (may have precipitate, mix well.) (4.0 µL) /sample
IVT Labeling NTP Mix (12.0 µL) /sample
IVT Labeling Enzyme Mix (4.0 µL) /sample

1. Mix components. Spin briefly.
2. Add 20.0 µL IVT MasterMix to each sample. Mix by pipetting.
3. 37 ºC for 16 hours in thermalcycler.
4. Clean-up cRNA with cRNA Clean-Up Module.

Clean-Up of Biotinylated cRNA with cRNA Clean-Up Module

Necessary Components:
cRNA Binding Buffer
cRNA Wash Buffer (ethanol added by MCF)
cRNA clean-up column (stored at 4°C in deli fridge)
Collection Tube (labeled with sample name on side)
DEPC H20
100% Ethanol
80% Ethanol
2 clean, labeled 1.5mL tubes

1. Transfer cRNA samples into clean, labeled 1.5mL tubes for clean-up.
2. Add 60.0 µL to each sample. Vortex to mix and spin down.
3. Add 350.0 µL cRNA Binding Buffer to each sample. Vortex and spin down.
4. Add 250.0 µL 100% ETOH to each sample. Mix by pipetting.
5. Add cRNA-Binding Buffer-ETOH mix onto column (it all fits).
6. Spin at 14,000rpm on “Short Spin” setting (30 seconds).
7. Place columns into clean, labeled collection tube. Discard previous tubes with eluate.
8. Add 500.0 µL cRNA Wash Buffer to each column.
9. Spin at 14K for “Short Spin”.
10. Discard eluate in sink but replace column into collection tube.
11. Add 500.0 µL 80% ETOH onto column.
12. “Short Spin” at 14K.
13. Discard eluate and replace columns into collection tube again.
14. Spin 14K for 5 minutes to dry column.
15. Put column into clean, labeled 1.5mL tube. (This tube should have the request number, the term “IVT” and the sample name on top. On the side put the PI last name and the date.)
16. Pipet 11.0 µL DEPC H20 onto center of column.
17. Spin 14K for 1 minute.
18. Add another 10.0 µL DEPC H20 onto center of column.
19. Spin 14K for 1 minute.
20. Measure volume of eluate. Record the volume in your lab notebook.
21. NanoDrop samples. Dilute with DEPC H20 if you need to, to get concentration into read-able range for NanoDrop (under 3000_g).
22. Make a dilution of the IVT samples for testing on the Agilent.

Perform calculations to determine adjusted cRNA amount. Enter the total µg into MAID. Perform calculations for Fragmentation step and record the volumes in your lab notebook. Calculations are done with the IVT NanoDrop adjusted cRNA concentrations acquired with the formula provided by Affy.

Fragmentation:

cRNA (X uL) 15ug
DEPC H20(variable) (32-x) µL
5X Fragmentation Buffer 8.0 µL
40.0 uL
1. Add 8.0 µL 5X Frag. Buffer to each empty 1.5mL tube.
2. Add the required amount of water to each tube. This amount will depend on the concentration of cRNA- how much cRNA to make 15 µg of fragmented cRNA, then how much DEPC H20 to bring the final volume to 40.0 µL?
3. Add the required amount of cRNA to each tube.
4. Make sure the 1.5mL tube is clearly labeled FRAG so that you can tell it apart from the IVT cRNA tube.
5. Put the FRAG tube onto the 99°C heat block for 35 minutes.
6. After fragmentation put the tube on ice. Let it cool about 5 minutes on ice.
7. Spin in the MicroFuge at max. speed to get the condensation out of the lid.
8. Vortex and spin.
9. Test FRAG on NanoDrop. Needs to be 15 µg or more. (15 µg/40 µL=0.375 µg/µL or more)

Put the FRAG into hybridization cocktail and store at -20°C until you are ready to load it onto a chip.
 
Hybridization protocol HYBRIDIZATION COCKTAIL

Euk 20X must be heated 5 minutes before using it. Vortex before using and spin down. Put on ice when you’re finished with it but not before adding it to MasterMix.
(multiply all by 3.5) MasterMix components

2X Hybe Buffer 150.0 µL 525.0 µL
DEPC H20 54.0 µL (to bring volume up to 300.0µL) 189.0 µL
100% DMSO 30.0 µL 105.0 µL
Eukaryotic 20X Control *** 15.0 µL 52.5 µL
Oligo B2 5.0 µL 17.5 µL
Herring Sperm DNA 3.0 µL 10.5 µL
BSA 3.0 µL 10.5 µL
cRNAfragmented 40.0 µL (if you retrieve all from NanoDrop) you have these separate

1. Keep cRNA in tubes labeled FRAG.
2. Prepare MasterMix in 15mL corning tube.
3. Mix after each component by pipetting several times.
4. Add 260.0 µL of the Hybe MasterMix to each fragmented sample. You total volume should be 300.0 µL.
5. If you are not hybridizing right away, store the cRNA-HybeCocktail mix in the -20°C until you’re ready to use it.

HYBRIDIZATION

Thaw Hybe Cocktails if they are frozen before heating. Pull arrays out of 4°C Deli Fridge 30 minutes before you want to load them. They must be at room temperature before you pierce the septa to prevent leakage.

1. Put tube hats on to prevent popping open at high heat.
2. Place the tubes into the 95°C heating block for 5 minutes.
3. Move the tubes to the 45°C heating block, again for 5 minutes.
4. Spin in MicroFuge at max. speed for 5 minutes.
5. Load 250.0 µL into each array.
6. Put in Hybe Oven, set at 60rpm and 45°C for 16 hours.

After 16 hours of hybridization, remove the hybridization cocktail from the probe array and fill the probe array completely with the appropriate volume of Non-Stringent Wash Buffer (Wash Buffer A)

Wash Buffer A: Non-Stringent Wash Buffer

(6X SSPE, 0.01% Tween-20)
For 1,000 mL:
300 mL of 20X SSPE
1.0 mL of 10% Tween-20
699 mL of water
Filter through a 0.2 μm filter
EukGE-WS2v5*
Midi_euk2*
10 cycles of 2 mixes/cycle with Wash
Buffer A at 30°C 6 cycles of 15 mixes/cycle with
Wash Buffer B at 50°C
Stain the probe array for 5 minutes in SAPE solution at 35°C
10 cycles of 4 mixes/cycle with Wash Buffer A at 30°C
Stain the probe array for 5 minutes in antibody solution at 35°C
Stain the probe array for 5 minutes in SAPE solution at 35°C
15 cycles of 4 mixes/cycle with Wash Buffer A at 35°C. The holding temperature is 25°C
Scan protocol [Scanner]
Pixel Size 1.56
hardware: GeneChip Scanner 3000
Affymetrix® Microarray Suite
Filter 570
Pixel Size 1.56
Scanner ID 50101490
Number of Scans 1
Scanner Type M10
As affymetrix protocol.
Description Lhx8 is a homeobox gene expressed in oocytes
Data processing Data in value is raw data (Mas5 scaled) unaltered by normalizations.
 
Submission date Mar 28, 2007
Last update date Aug 28, 2018
Contact name Aleksandar Rajkovic
E-mail(s) rajkovic@bcm.edu
Organization name Baylor College of Medicine
Street address One Baylor Plaza
City Houston
State/province TX
ZIP/Postal code 77030
Country USA
 
Platform ID GPL1261
Series (2)
GSE7774 Transcriptional changes in Lhx8 Null newborn mouse ovaries
GSE11897 Lim Homeobox Gene, LHX8, Is Essential for Mouse Oocyte Differentiation and Survival
Relations
Reanalyzed by GSE119085

Data table header descriptions
ID_REF
VALUE Mas5 scaled raw signal
ABS_CALL Affymetrix present/absent flag

Data table
ID_REF VALUE ABS_CALL
AFFX-BioB-5_at 689.8 P
AFFX-BioB-M_at 1054.2 P
AFFX-BioB-3_at 629 P
AFFX-BioC-5_at 2006 P
AFFX-BioC-3_at 2785.8 P
AFFX-BioDn-5_at 5025.7 P
AFFX-BioDn-3_at 8503.1 P
AFFX-CreX-5_at 25788.3 P
AFFX-CreX-3_at 30366.8 P
AFFX-DapX-5_at 25 P
AFFX-DapX-M_at 16.4 A
AFFX-DapX-3_at 10.5 A
AFFX-LysX-5_at 9.9 A
AFFX-LysX-M_at 2.1 A
AFFX-LysX-3_at 14.1 A
AFFX-PheX-5_at 1.2 A
AFFX-PheX-M_at 1.4 A
AFFX-PheX-3_at 10.5 A
AFFX-ThrX-5_at 5.3 A
AFFX-ThrX-M_at 12.5 A

Total number of rows: 45101

Table truncated, full table size 826 Kbytes.




Supplementary file Size Download File type/resource
GSM178086.CEL.gz 5.9 Mb (ftp)(http) CEL
Processed data included within Sample table

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