|
| Status |
Public on Dec 02, 2014 |
| Title |
Blood_control 3 |
| Sample type |
RNA |
| |
|
| Source name |
healthy control
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: peripheral blood mononuclear cells
|
| Treatment protocol |
Buffer RPE is supplied as a concentrate. Before using for the first time, add 4 volumes of ethanol (96–100%) as indicated on the bottle to obtain a working solution.
|
| Growth protocol |
Lyse cells directly in a culture dish by adding 1 ml of TRIzol Reagent to a 3.5 cm diameter dish, and passing the cell lysate several times through a pipette. The amount of TRIzol Reagent added is based on the area of the culture dish (1 ml per 10 cm2) and not on the number of cells present.Pellet cells by centrifugation. Lyse cells in TRIzol Reagent by repetitive pipetting. Use 1 ml of the reagent per 5-10 × 106 of animal, plant or yeast cells, or per 1 × 107 bacterial cells. Washing cells before addition of TRIzol Reagent should be avoided as this increases the possibility of mRNA degradation. Disruption of some yeast and bacterial cells may require the use of a homogenizer.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was harvested using TRIzol (Invitrogen) and miRNeasy mini kit (QIAGEN) according to manufacturer’s instructions.
|
| Label |
cy3
|
| 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 2×Hybridization buffer 90 Nuclease-free Buffer 77.5 Total 180 2. Incubate at 95°C 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 “securely” 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.
|
| 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.
|
| Description |
Healthy controls < 18 years
|
| 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 |
Jan 31, 2014 |
| Last update date |
Dec 02, 2014 |
| Contact name |
Fuquan Zhang |
| E-mail(s) |
zfq99@hotmail.com
|
| Organization name |
Wuxi mental health center
|
| Street address |
156 Qianrong Rd
|
| City |
Wuxi |
| ZIP/Postal code |
214151 |
| Country |
China |
| |
|
| Platform ID |
GPL16016 |
| Series (2) |
| GSE54578 |
miRNA expression in blood of 15 schizophrenia patients and 15 healthy controls |
| GSE54914 |
Expression in blood of schizophrenia patients and healthy controls |
|
