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Sample GSM143024 Query DataSets for GSM143024
Status Public on Mar 21, 2009
Title KSUAP-DM mReissners Membrane I24hrA
Sample type RNA
 
Source name Reissner's Membrane incubated for 24 hours
Organism Mus musculus
Characteristics Tissue: Reissner's Membrane
Animal: Mouse
Age: adult
Biomaterial provider Cellular Biophysics laboratory, Kansas State University
Treatment protocol Reissner's Membrane explants incubated for 24 hours
Growth protocol Native tissue; no proliferation in organ culture
Extracted molecule total RNA
Extraction protocol RNA was extracted using a RNeasy Microkit following the manufacturer's protocol (no. 74004, Qiagen; Valencia, CA)
Label Affymetrix Small Sample Labeling Protocol vII (2xIVT)
Label protocol First Cycle of Amplification
Step 1. First cycle, first strand cDNA synthesis
Note: Use thermal cycler with a heated lid for incubations in this step. Use the 4C hold for the addition of reagents. 70C 6 minutes
4C hold
42C 60 minutes
70C 10 minutes
4C hold
a. Mix 1ul of total RNA (50ng/ul) with 1ul of 5uM T7(dT)24 in 0.2ul PCR tube and incubate at 70C in thermal cycler with heated lid for 6 min.
(5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTTTTTTTTTTTTTTTT-3’)
b. Cool to 4C for 2 min.
c. Spin briefly.
d. Add 3ul of RT-Premix-1 master mix using the following components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least four reactions to avoid pipetting small volumes.
1 rxn
DEPC treated water 0.375ul
5x first strand buffer 1ul
DTT, 0.1M 0.5ul
dNTP mix, 10mM 0.375ul
Rnase inhibitor, 40 U/ul 0.25ul
SuperScript II, 200 U/ul 0.5ul
Total Volume 3ul
e. Incubated at 42C 60 minutes.
f. Heat sample at 70C for 10 minutes to inactivate SuperScript II.
g. Spin briefly and cool sample to 4C.
Step 2. First cycle, second strand cDNA synthesis
Note: Use thermal cycler without heated lid for incubations in this step. Use 4C hold for the addition of reagents.
16C 120 minutes
4C hold
16C 10 minutes
4C hold
a Prepare the SS-Premix-1 as follows using the components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least four reactions to avoid pipetting small volumes.
1 rxn
DEPC treated water 22.75ul
5x second strand buffer 7.5ul
dNTP mix, 10mM 0.75ul
DNA ligase, E. coli, 10U/ul 0.25ul
DNA polymerase I, E. coli 10U/ul 1ul
Rnase H, 2U/ul 0.25ul
Total Volume 32.5ul
b. Add 32.5ul of the SS-Premix-1 to each first strand reaction making a final volume of 37.5ul.
c. Mix by pipetting and spin briefly.
d. Incubate the samples at 16C for 2 hours.
e. Add 1ul of T4 DNA polymerase (5U/ul) to the reaction and mix by pipetting.
f. Continue incubation at 16C for 10 minutes.
Step 3. First cycle, double-stranded cDNA cleanup by ethanol precipitation.
a. Transfer the reaction to a 1.5 ml centrifuge tube.
b. Add 80ul of DEPC treated water to dilute reaction.
c. Add 2ul of glycogen (5 mg/ml), 0.6 volumes (72ul) of 5M NH4OAc, and 2.5 volumes (480ul) of cold absolute ethanol. Mix by pipetting.
d. Precipitate the double-stranded cDNA at -20C for 2 hours.
e. Centrifuge at 14,000rpm for 20 minutes at 4C.
f. Wash pellet with 800ul of 70% cold ethanol.
g. Centrifuge at 14,000rpm for 5 minutes at 4C.
h. Remove ethanol and dry pellet in speed vacuum for 10 minutes.
Step 4. First cycle, IVT for cRNA amplification using MEGAscript T7 kit (Ambion).
a. At room temperature, add the following reagents to the dried double-stranded cDNA pellet, in the order indicated.

1rxn
DEPC treated water 4ul
Premixed NTPs, 18.75mM each 4ul
10x reaction buffer 1ul
10x enzyme mix 1ul
Total Volume 10ul
b. Mix well by pipetting. Spin briefly.
c. Incubate at 37C for 5 hours mixing every 30 minutes.
Step 5. First cycle, cRNA cleanup with Qiagen RNeasy columns.
a. Add 90ul of Rnase-free water to sample.
b. Follow the RNeasy Mini Protocol for RNA Cleanup as directed in the RNeasy Mini Kit handbook.
c. In the last step of the purification, elute the cRNA sample with 30ul of Rnase-free water first, and follow with an additional 20ul of water for the second elution.
d. Determine the cRNA yield by conducting a 1/10 dilution in water and measuring the absorbance at 260nm.
e. Transfer 400ng of cRNA to a new 1.5ml tube and use speed vacuum to concentrate sample to 4ul. Avoid drying the sample completely. Note: If the yield is less that 400ng, use the entire sample for the second cycle of cDNA synthesis.
Second Cycle of Amplification and Labeling
Step 6. Second cycle, first strand cDNA synthesis.
Note: Use heating blocks for incubation in this step.
a. Add random primers to the cRNA sample and mix well.
cRNA, 400ng 4ul
Random primers, 0.2 ug/ul 1ul
Total Volume 5ul
b. Incubate at 70C for 10 minutes to denature cRNA.
c. Cool sample on ice for 2 minutes.
d. Prepare the RT-Premix-2 as follows using the components from the SuperScript Choice System (Invitrogen). It is recommended to assemble the master mix for at least two reactions to avoid pipetting small volumes.

1rxn
5x first strand buffer 2ul
DTT, 0.1M 1ul
dNTP mix, 10mM 0.5ul
RNase Inhibitor, 40 U/ul 0.5ul
SuperScript II, 200 U/ul 1ul
Total Volume 5ul
e. Add 5ul of the RT-Premix-2 to the denatured RNA and primer mixture, making a final volume 10ul. Mix well by pipetting. Spin briefly.
f. Incubate the sample at 42C for 1 hour. Spin briefly.
g. To remove the RNA template, add 1ul of RNase H (2 U/ul) and incubate the sample at 37C for 20 minutes.
h. Heat the sample at 95C for 5 minutes to inactivate RNase H.
i. Cool sample on ice 2 minutes. Spin briefly.
Step 7. Second cycle, second strand cDNA synthesis
Note: Transfer sample to a 0.2 ml PCR tube and perform the incubations in a thermal cycler without a heated lid. Use the 4C hold to add reagents.
70C 6 minutes
4C hold
16C 120 minutes
4C hold
16C 10 minutes
4C hold
a. Add 2.0ul of the 5uM T7(dT)24 promoter primer to the chilled sample from the previous step.
b. Mix well. Spin briefly.
c. Incubate sample at 70C for 6 minutes.
d. Cool the sample to 4C and spin briefly.
e. Prepare the SS-Premix-2 as follows using the components from the SuperScript Choice System (Invitrogen).
1rxn
DEPC treated water 43.5ul
5x second strand buffer 15ul
dNTP mix, 10mM 1.5ul
DNA polymerase I, E. coli, 10 U/ul 2ul
Total Volume 62ul
f. Add 62ul of the SS-Premix-2 to the sample making a total volume of 75ul. Mix well by pipetting. Spin briefly.
g. Incubate at 16C for 2 hours.
h. Add 2ul of T4 DNA polymerase (5 U/ul) to the reaction. Mix well.
i. Incubate at 16C for 10 minutes.
Step 8. Second cycle, double-strand cDNA cleanup by ethanol precipitation.
a. Add 2ul of 5 mg/ml glycogen, 0.6 volume 5M NH4OAc, and 2.5 volumes of cold absolute ethanol.
b. Mix well by pipetting.
c. Precipitate the double-stranded cDNA at -20C for 30 minutes.
d. Centrifuge the sample at 14,000 rpm for 20 minutes at 4C.
e. Carefully remove supernatant and wash the cDNA pellet by adding 800ul of 70% cold ethanol.
f. Centrifuge the tube at 14,000 rpm for 5 minutes at 4C.
g. Carefully remove the ethanol. Dry pellet in a speed vacuum for 10 minutes.
h. Store dry pellet at –20C or proceed to next step.
Step 9. Second cycle, IVT for cRNA amplification and labeling with ENZO BioArray High Yield RNA Transcription
Labeling Kit (Affymetrix) or GeneChip Expression 3’-Amplification IVT Labeling kit (Affymetrix).
a. At room temperature, add the following reagents to the dried double-stranded cDNA pellet.
Enzo BioArray High Yield RNA Transcription Labeling kit
1rxn
DEPC treated water 22ul
10X HY reaction buffer 4ul
10X Biotin labeled ribonucleotides 4ul
10X DTT 4ul
10X RNase inhibitor mix 4ul
20X T7 RNA polymerase 2ul
Total Volume 40ul
1. Mix the components well after adding each reagent; especially after adding water, ensuring the cDNA
pellet is completely dissolved.
2. Incubate at 37C for 5 hours mixing every 30 minutes.
GeneChip Expression 3’-Amplification IVT Labeling Kit
1rxn
DEPC treated water 20ul
10x IVT Labeling Buffer 4ul
IVT Labeling NTP Mix 12ul
IVT Labeling Enzyme Mix 4ul
Total Volume 40ul
1. Mix components well after adding each reagent, especially after adding water, ensuring the cDNA pellet
is completely dissolved.
2. Incubate at 37ºC for 16 hours in a hybridization oven or thermal cycler with a heated lid to prevent
condensation.
Step 10. Second cycle, labeled cRNA target cleanup with Qia
 
Hybridization protocol GENECHIP HYBRIDIZATION PROTOCOL:
Hybridization cocktail composition: (for standard array format) Note: 10% DMSO is added for targets
Prepared using the GeneChip
Expression 3’-Amplificaton IVT
Labeling Protocol.
30ul fragmented cRNA 15ug (0.05ug/ul final conc.)
5ul control Biotin labeled oligo B2 (3nM) (Affymetrix) (50pM final conc.)
15ul 20x Eukaryotic Hybridization spike controls
(bioB, bioC, BioD, cre) (Affymetrix) (1.5, 5, 25 and 100pM final conc.
respectively)
3ul Herring Sperm DNA (10mg/ml)(blocking reagent) (0.1mg/ml final conc.)
3ul Acetylated BSA (50mg/ml)(blocking reagent) (0.5mg/ml final conc.)
150ul 2x Hybridization Buffer
(Final 1x conc. is 100mM MES {12X stock: 70.4g MES free-acid monohydrate, 193.3g MES
sodium Salt/L pH 6.6}, 1M [Na+], 20mM EDTA, 0.01% Tween 20)
- DEPC H2O to 300ul.
- Denatured at 99C 5min prior to applying to GeneChip
Hybridization Conditions:
- GeneChip is prehybridized with 200ul 1x Hybridization buffer (see above) at 45C 10min at 60rpm
- 200ul denatured Hybridization cocktail is applied to GeneChip
- GeneChip is hybridized 16 hr at 45C and 60rpm in GeneChip Hybridization Oven 640.
Washing and Staining: Automated process using the GeneChip Fluidics Station 400 with the Standard
Array Format.
Enzo BioArray High Yield RNA Transcript Labeling Washing and Staining Protocol
Post Hyb wash #1 - 10 cycles of 2 mixes/ cycle with wash buffer A at 25C - non-stringent
(6xSSPE, 0.01% Tween 20)
Post Hyb wash #2 - 4 cycles of 15 mixes/ cycle with wash buffer B at 50C - stringent (100mM
MES, 0.1M [Na+], 0.01% Tween 20)
1st Stain - 10 min. at 25C (SAPE Stain: 300ul 2x MES stain buffer {41.7ml 12x MES,
92.5ml 5M NaCl, 2.5ml 10% Tween 20/ 250ml total vol. in DEPC H2O},
24ul 50mg/ml acetylated BSA, 6ul 1mg/ml Streptavidin-R-Phycoerythrin
conjugate, 270ul DEPC H2O, 600ul total vol.)
Post Stain Wash - 10 cycles of 4 mixes/cycle with wash buffer A at 25C
2nd Stain - 10 min. at 25C (Antibody Stain: 300ul 2x MES stain buffer {see above},
24ul 50 mg/ml acetylated BSA, 6ul 10mg/ml normal goat IgG,
3.6ul 0.5mg/ml biotinylated anti-streptavidine antibody, 266.4ul DEPC H2O,
600ul total vol.)
3rd Stain - 10 min at 25C (SAPE stain: see above, 600ul total vol.)
Final Wash - 15 cycles of 4 mixes/cycle with wash buffer A at 30C. The GeneChip remains
filled with wash buffer A for the scanning procedure.
GeneChip Expression 3’-Amplification IVT Labeling Washing and Staining Protocol
Post Hyb wash #1 - 10 cycles of 2 mixes/ cycle with wash buffer A at 30ºC - non-stringent
(6xSSPE, 0.01% Tween 20)
Post Hyb wash #2 - 6 cycles of 15 mixes/ cycle with wash buffer B at 50ºC - stringent (100mM
MES, 0.1M [Na+], 0.01% Tween 20)
1st Stain - 5 min. at 30ºC (SAPE Stain: 300ul 2x MES stain buffer {41.7ml 12x MES,
92.5ml 5M NaCl, 2.5ml 10% Tween 20/ 250ml total vol. in DEPC H2O},
24ul 50mg/ml acetylated BSA, 6ul 1mg/ml Streptavidin-R-Phycoerythrin
conjugate, 270ul DEPC H2O, 600ul total vol.)
Post Stain Wash - 10 cycles of 4 mixes/cycle with wash buffer A at 30ºC
2nd Stain - 5 min. at 35ºC (Antibody Stain: 300ul 2x MES stain buffer {see above},
24ul 50 mg/ml acetylated BSA, 6ul 10mg/ml normal goat IgG,
3.6ul 0.5mg/ml biotinylated anti-streptavidine antibody, 266.4ul DEPC H2O,
600ul total vol.)
3rd Stain - 5 min at 35ºC (SAPE stain: see above, 600ul total vol.)
Final Wash - 15 cycles of 4 mixes/cycle with wash buffer A at 35ºC. The GeneChip remains
filled with wash buffer A for the scanning procedure.
Scan protocol Scanning: Automated process conducted using the Agilent GeneArray Scanner.
- 2x scans are conducted for the Enzo protocol and 1x scans are conducted for the GeneChip
Expression protocol as follows:
Pixel value = 3um
Wavelength = 570nm
Description Absolute and comparison analysis are conducted using the following settings:
Scaling - All Probe Sets: Target Signal = 500
Normalization – User Defined: Scale Factor = 1
Data processing Data was generated and calculated in GCOS1.2
 
Submission date Oct 28, 2006
Last update date Aug 28, 2018
Contact name Daniel Marcus
E-mail(s) marcus@vet.ksu.edu
Phone 785-532-4532
Organization name Kansas State University
Department Anatomy & Physiology
Lab Cellular Biophysics lab
Street address 228 Coles Hall
City Manhattan
State/province KS
ZIP/Postal code 66506
Country USA
 
Platform ID GPL1261
Series (1)
GSE6196 Mouse Reissner's Membrane Gene Expression studies
Relations
Reanalyzed by GSE119085

Data table header descriptions
ID_REF
VALUE Signal Intensity
ABS_CALL Quality calls - Present, Marginal, Absent calls
DETECTION P-VALUE Detection p-value

Data table
ID_REF VALUE ABS_CALL DETECTION P-VALUE
AFFX-BioB-5_at 345.9 P 0.000509
AFFX-BioB-M_at 417.2 P 0.000044
AFFX-BioB-3_at 280.7 P 0.000044
AFFX-BioC-5_at 911.5 P 0.000044
AFFX-BioC-3_at 1122 P 0.000052
AFFX-BioDn-5_at 1851.7 P 0.000044
AFFX-BioDn-3_at 3304.3 P 0.000044
AFFX-CreX-5_at 9486.2 P 0.000044
AFFX-CreX-3_at 12436.1 P 0.000044
AFFX-DapX-5_at 9.4 A 0.300606
AFFX-DapX-M_at 14 A 0.51489
AFFX-DapX-3_at 13.1 A 0.544591
AFFX-LysX-5_at 5.4 A 0.340661
AFFX-LysX-M_at 4.4 A 0.794268
AFFX-LysX-3_at 12.1 A 0.455413
AFFX-PheX-5_at 1 A 0.978107
AFFX-PheX-M_at 9.2 A 0.659339
AFFX-PheX-3_at 10.6 A 0.382599
AFFX-ThrX-5_at 13.6 A 0.455413
AFFX-ThrX-M_at 4.1 A 0.672935

Total number of rows: 45101

Table truncated, full table size 1211 Kbytes.




Supplementary file Size Download File type/resource
GSM143024.CEL.gz 6.1 Mb (ftp)(http) CEL

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