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Sample GSM230044 Query DataSets for GSM230044
Status Public on Dec 05, 2007
Title P493 +tet (low Myc)
Sample type RNA
 
Source name P493 cells treated with tetracycline for 72 hours
Organism Homo sapiens
Characteristics P493 cells treated with tetracycline for 72 hours
Extracted molecule total RNA
Extraction protocol Trizol according to manufacturer's instructions
Label Cy3
Label protocol 1. Assemble RNA Labeling Reaction:
(amounts are per sample to be tested)
1.2µL 10X Iglori Buffer
1.2mL 100% DMSO
1µL .105µM Bio B Primer
1µL .35µM Bio C Primer
2µL Cy-3 (250ng/µL)
1µL 20U/µL T4 RNA Ligase
Add 7.4µL Labeling Solution to 5µL of 10µg Total RNA
Incubate on ice for 2 hours.
2. After 2 hours of labeling, add 46µL of RNA precipitation solution:
(prepare immediately before use)
40µL DEPC Water
5µL 3M Sodium Acetate (pH5.2)
1µL Glycogen
Add 150µL of 100% ethanol and finger vortex. Place on ice for 10 minutes. Centrifuge for 10 minutes, max speed @ 4°C. Remove supernatant. Add 150µL of 70% ethanol. Finger vortex. Spin 5 minutes, max speed @ 4°C. Remove ethanol. Briefly spin again at max speed. Remove remaining ethanol with a p20 pipet. Let pellet air dry for 2-3 minutes. Remove supernatant. Resuspend pellet with 6µL DEPC water.
 
Hybridization protocol 1. Pipet 35µL of the prehyb solution into one hyb portal of the hyb cap.
Prehyb Solution: (prepare immediately before use)
Amount appropriate for one array
22.5µL 20X SSC
1.5µL 10% SDS
15µL 10X (2%) BSA
111µL DEPC H2O
Make sure there are no bubbles in the hyb chamber. Also take care that none of the prehyb solution comes out of the hyb portal on the other side hyb chamber. Securely tape the hyb portals with Scotch Tape. Place the hyb clamp assembly into a hyb tube fitted with a large kimwipe in such a way that the clamp assembly does not move. Place the hyb tube into a hybridization oven, and allow to rotate for a minimum of 1 hour at 37°C.
2. Prepare hybridization buffer:
(amounts are per sample)
17.5µL 2X Hyb Solution (7.5mL 1M Na2HPO4, 150mg BSA, 0.938g Ultrapure SDS, 1.88ml DEPC Water)
4.2µL DI Formamide
7.3µL DEPC Water
Add labeled RNA to 29µL of hybridization buffer.
3. Heat sample for 3 minutes at 95°C.
4. Remove prehyb solution from the hyb portals. Add 35µL of sample-hyb mix directly to hyb portals Make sure there are no bubble in the hyb chamber. Also take care that none of the hyb solution comes out of the hyb portal on the other side hyb chamber.
5. Securely tape the hyb portals with Scotch Tape. Place the hyb clamp assembly into a hyb tube fitted with a large kimwipe in such a way that the clamp assembly does not move. Place the hyb tube into a hybridization oven, and allow to rotate overnight at 37°C.
6. Remove hyb solution from the hyb portals. Quickly rinse the chambers with Wash #1 (2X SSC, 0.025% SDS). Remove Wash, and refill chambers with 35µL Wash #1. Incubate at room temp for 3 minutes.
7. Remove Wash #1, and quickly rinse chambers with 35µL Wash #2 (1.6X SSC). Remove Wash, and refill chambers with 35µL Wash #2. Incubate at room temp for 3 minutes. Repeat 2 more times.
8. Remove Wash #2, and quickly rinse chambers with 35µL of ice cold Wash #3 (0.8X SSC) in a cold room. Remove Wash, and refill chambers with 35µL Wash #3. Incubate at 4°C room temp for 3 minutes. Repeat 2 more times. Remove all wash solution.
9. Remove array from hyb clamp, and remove hyb cap.
10. Add 1 or 2 drops of Imaging Solution (from Combimatrix) to array.
11. Carefully clean hyb cover slips with 70% EtOH.
12. Using forceps, place cover slip over semiconductor portion of array, with the white (rough) strips down, making sure no bubbles are trapped. Blot excess solution from edges of cover slip.
13. Keep in the dark until ready to scan.
Scan protocol Scan with GenePix 4000B microarray scanner.
Description N/A
Data processing Hybridization signals extracted using Combimatrix Microarray Imager software. VALUE column represents median-normalized, background-subtracted hybridization signals.
 
Submission date Sep 17, 2007
Last update date Aug 14, 2011
Contact name Tsung-Cheng Chang
Organization name Johns Hopkins University School of Medicine
Street address 733 N. Broadway
City Baltimore
State/province MD
ZIP/Postal code 21205
Country USA
 
Platform ID GPL5847
Series (2)
GSE9070 Identification of Myc-regulated microRNAs in human P493 cells
GSE9129 Identification of Myc-regulated microRNAs: human and mouse

Data table header descriptions
ID_REF
VALUE Normalized, background-subtracted hybridization signal

Data table
ID_REF VALUE
hsa|miR-200c|as| 167.8077479
hsa|miR-125a|as| 170.2604799
hsa|miR-199a|as| 170.8736629
hsa|miR-346|as| 171.8956346
hsa|miR-32|as| 172.5088176
LR|hsa-mir-425|3-Prime|as| 172.7132119
hsa|miR-425|as| 174.5527609
mghv|miR-M1-2|as| 177.2098873
LR|hsa-mir-324|5-Prime|as| 178.4362533
hsa|miR-452|as| 181.0933796
LR|hsa-mir-212|3-Prime|as| 181.2977739
hcmv|miR-US25-2-5p|as| 181.7065626
hsa|Actb|AK125561|_1649_1673|as| 182.3197456
hsa|miR-453|as| 182.9329286
hsa|miR-370|as| 186.6120266
hsa|miR-198|as| 187.8383926
hsa|miR-140|as| 191.1087019
hsa|miR-133a|as| 194.1746169
hsa|miR-520d|as| 196.2185603
LR|hsa-mir-139|5-Prime|as| 204.598728

Total number of rows: 1161

Table truncated, full table size 28 Kbytes.




Supplementary file Size Download File type/resource
GSM230044.txt.gz 6.4 Kb (ftp)(http) TXT
Processed data included within Sample table

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