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| Status |
Public on Aug 05, 2023 |
| Title |
TAT+AEA_ A_BR1 |
| Sample type |
RNA |
| |
|
| Source name |
Human Brain
|
| Organism |
Homo sapiens |
| Characteristics |
treatment: miRNA expression with 24 hr TAT+AEA treatment
|
| Treatment protocol |
Once the cells attained 80% confluency, they were treated with HIV-1 TAT recombinant protein, AEA and TAT+AEA for 24 hours.
|
| Growth protocol |
Normal Human Astrocyte (Cat. No: CC-2565 ) from Lonza had been cultured with Lonza AGMTM Astrocyte Growth Medium Bullet KitTM (basal medium and Single QuotesTM Kit; Cat. No: CC-3186) for cell specific growth. Cells were seeded into a T75 culture flask and allowed to grow in CO2 incubator for a week to attain 80% confluency. Cells were split in 6 well plates for treatment.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Treated astrocyte cells were harvested by using TRIzol™ Reagent (Cat. No: 15596018) InvitrogenTM. By using 200 μl of chloroform reagent, phase separation was obtained after centrifugation at 12000 rpm for 15 minutes at 4 degree C. The clear upper aqueous phase was collected and the RNA was precipitated by using 500 μl ice cold isopropanol. Isopropanol is removed then by using 70% ethanol at 12000 rpm for 10 minutes. Then the precipitate was air dried and dissolved in RNase free water.
|
| Label |
Cy3
|
| Label protocol |
The miRNA labeling was performed using miRNA Complete Labeling and Hyb Kit (Agilent Technologies, Part Number: 5190-0456).The total RNA sample was diluted to 100ng/ul in nuclease free water. About 200ng of total RNA was dephosphorylated using Calf Intestinal Alkaline Phosphatase (CIP) master mix (Agilent Technologies, Part Number: 5190-0456) by incubating at 37oC for 30 minutes. The dephosphorylated miRNA sample was denatured by adding Dimethyl Sulfoxide (DMSO) and heating at 100 oC for 10 minutes and transferred to ice-water bath. The Ligation master mix (Agilent Technologies, Part Number: 5190-0456) containing Cyanine 3-pCp was added to the denatured miRNA sample and incubated at 16oC for 2 hours. The Cyanine 3-pCp labeled miRNA sample was dried completely in the vacuum concentrator (Eppendorf, Concentrator Plus, Catalog Number 5305000) at 45oC for 2 hour.
|
| |
|
| Hybridization protocol |
The dried sample was resuspended in nuclease free water and mixed with Hybridization Mix containing blocking solution (Agilent Technologies, Part Number: 5190-0456) and Hi-RPM Hybridization Buffer (Agilent Technologies, Part Number: 5190-0456) and incubated at 100oC for 5 minutes followed by snap chill on ice for 5 minutes. The samples were hybridized on the Human miRNA 8x60K Arrays. The hybridization was carried out at 55°C for 20 hours. After hybridization, the slides were washed using Gene Expression Wash Buffer1 (Agilent Technologies, Part Number 5188-5325) at room temperature for 5 minutes and Gene Expression Wash Buffer 2 (Agilent Technologies, Part Number 5188-5326) at 37oC for 5 minutes.
|
| Scan protocol |
The microarray slide was scanned on a G2600D scanner (Agilent Technologies)
|
| Data processing |
Images were quantified using Feature Extraction Software ( Agilent). Feature extracted raw data was analyzed using GeneSpring GX software from Agilent. Normalization of the data was done in GeneSpring GX using the 50th percentile shift miRNA based.
|
| |
|
| Submission date |
Jul 29, 2022 |
| Last update date |
Aug 05, 2023 |
| Contact name |
Genotypic technology |
| E-mail(s) |
sudha.rao@genotypic.co.in
|
| Organization name |
Genotypic Technology
|
| Street address |
259, Apoorva 4th cross,80 feet Road,RMV 2ND STAGE
|
| City |
Bangalore |
| State/province |
Karnataka |
| ZIP/Postal code |
560094 |
| Country |
India |
| |
|
| Platform ID |
GPL32522 |
| Series (1) |
| GSE210095 |
Effect of endocannabinoid AEA on HIV1 TAT activated Normal Human Primary Astrocytes |
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