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| Status |
Public on Sep 14, 2017 |
| Title |
patient 15 ZFN |
| Sample type |
RNA |
| |
|
| Source name |
plasma necrosis
|
| Organism |
Homo sapiens |
| Characteristics |
individual: patient 15
disease state: necrosis
sample type: serum
|
| Extracted molecule |
total RNA |
| Extraction protocol |
WHOLE BLOOD Add 0.2 ml of whole blood or plasma to 0.75 ml of TRI Reagent BD supplemented with 20 ul of 5 N acetic acid per 0.2 ml of whole blood or plasma
|
| Label |
Hy3
|
| Label protocol |
miRCURYTM Array Power Labeling kit (Cat #208032-A, Exiqon)
|
| |
|
| Hybridization protocol |
1. Prepare the hybridization mix in a PCR tube according to the table below: Components Amount(ul) Labeled sample 12.5 μlHybridization buffer 90 Nuclease-free Buffer 77.5 Total 180 2. Incubate at 95℃ for 2 min. During the incubation the target preparation should be protected from light. 3. Leave on ice for at least 2 min. and up to 15min. Briefly spin the reaction after ice incubation. 4. Take cover slide out of the package. 5. Carefully laid the miRCURYTM Array on top of the assembly of cover slide and spacer, where the printed side of array should be facing toward the cover slide, to form a hybridization assembly. The printed side of array is on the side of the label. 6. Insert the hybridization assembly into a (3.1 x 9cm) heat-shrank hybridization (hyb.) bag. Sample loading side of the hybridization assembly should face the opening side of film bag. Lower the assembly to the end of the bag (Assembly viewed from the other side). 7. Clip the bag with a clipper ecurely?from the opening end. Immerse the assembly to the rim of the slide in 95℃ hot water swiftly (approximately 2~5 sec). Do not dip the assembly too far into the water to avoid water leaking into the assembly. The hyb. bag will shrink and tightly wrap around the assembly. 8. Remove the assembly from water and wipe off the water from the assembly. Trim off the excess film from the top of the assembly. Keep the assembly in a 50℃ oven for at least 10 minutes. 9. Load the180ul target hybridization mix into the assembly through the sample loading opening. Fill up the hybridization space with 1X Hybridization buffer till the liquid level reaches to approximately 5 mm (0.5cm) away from the top rim. 10. Cut the second Hyb. Bag to approximately one-half of its original length. 11. Keep the assembly vertical and slip on the shortened hyb. bag from step 11 all the way until the assembly reaches the end of the hyb. bag. 12. Use a pair of forceps to hold onto the top of the assembly. Immerse the entire assembly into 95℃ hot water with the assembly in upright position. 13. Place the hybridization assembly into a 56℃ oven and set it on a rotator. Remember to rotate the assembly at 2 rotations per minute (RPM) for overnight. 14. After hybridization, disassembled the hybridization assembly, and wash the slides at 56℃ for 2min. using Wash buffer A. 15. Wash briefly at room temperature in Wash buffer B. 16. Wash for 2min. at room temperature in Wash buffer B. 17. Wash for 2min. at room temperature in Wash buffer C. 18. Wash briefly in water. 19. Dry the slides by centrifugation for 5 min. at 200* g(1000rpm). Scan slides immediately after drying.
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| Scan protocol |
Scanning is performed with the Axon GenePix 4000B microarray scanner. GenePix pro V6.0 is used to read the raw intensity of the image.
|
| Data processing |
a) The intensity of green signal is calculated after background subtraction and four Replicated spots of each probe on the same slide have been averaged. b) We use Median Normalization Method to obtain “Normalized Data”, Normalized Data= (Foreground-Background)/median, the median is 50 percent quantile of microRNA intensity which is larger than 50 in all samples after background correction. c) After normalization, the statistical significance of differentially expressed miRNA was analyzed by T-test.
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| |
|
| Submission date |
Nov 07, 2016 |
| Last update date |
Sep 14, 2017 |
| Contact name |
zhang ying |
| Organization name |
Medical Center of Hip, Luoyang Orthopedic-Traumatological Hospital (Orthopaedic Hospital of Henan Province)
|
| Street address |
No. 82, South Qiming Road
|
| City |
Luoyang |
| State/province |
Henan |
| ZIP/Postal code |
471002 |
| Country |
China |
| |
|
| Platform ID |
GPL21439 |
| Series (1) |
| GSE89587 |
MicroRNAs expression different in Serum between Trauma-induced osteonecrosis of the femoral head (TIONFH) and healing patients |
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