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| Status |
Public on Dec 27, 2021 |
| Title |
Cont_drug.gpr |
| Sample type |
RNA |
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| Source name |
GDC-0152+U937 cells
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| Organism |
Homo sapiens |
| Characteristics |
rna fraction: exosome
cell line: U937
gender: male
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| Treatment protocol |
The human acute monocytic leukemia cell line, U937, was purchased from Bena Culture Collection (Beijing, China) and stored in our laboratory. U937 cells were cultured in RPMI-1640 (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) with 10% fetal bovine serum (Biological Industries, Beit, HaEmek, Israel), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Gibco/Thermo Fisher Scientific, Waltham, MA, USA). The cells were grown in a 37°C incubator with 5% CO2. A recombinant lentivirus vector–mediated SQSTM1 gene (LV-SQSTM1-RNAi) and an empty recombinant adenovirus vector (Hu6-MCS-CMV-EGFP) were constructed. U937 cells were placed in a six-well plate. Polybrene (Gene, Shanghai, China) was used for the transfection. The transfection system included 1.8 mL of RPMI-1640 with 10% fetal bovine serum, 10 μL of LV-SQSTM1-RNAi or Hu6-MCS-CMV-EGFP and 0.9 μL of polybrene. After transfection for 24 h, the cell suspension was collected and centrifuged at 800 rpm for 5 min, the supernatant with lentivirus discarded, and 2 mL of RPMI-1640 with 10% fetal bovine serum then added.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using TRIzol (Invitrogen) . RNA quality and quantity was measured by using nanodrop spectrophotometer (ND-1000, Nanodrop Technologies) and RNA Integrity was determined by gel electrophoresis. section stained by May-Giemsa. Careful attention was paid to the mi
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| Label |
Hy3
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| Label protocol |
After quality control, the miRCURY™ Hy3™/Hy5™ Power labeling kit (Exiqon, Vedbaek, Denmark) was used according to the manufacturer’s guideline for miRNA labelling by following steps: a, 1μLRNA in 2.0 μL of water was combined with 1.0 μL of CIP buffer and CIP (Exiqon). The mixture was incubated for 30 min at 37°C. b, The Reaction was terminated by incubation for 5 min at 95°C. Then 3.0 μL of labeling buffer, 1.5 μL of fluorescent label (Hy3TM), 2.0 μL of DMSO, 2.0 μL of labeling enzyme were added into the mixture.The labeling reaction was incubated for 1 h at 16°C c, Terminated by incubation for 15 min at 65°C.
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| Hybridization protocol |
After stopping the labeling procedure, the Hy3™-labeled samples were hybridized on the miRCURYTM LNA Array (v.19.0) (Exiqon) according to array manual. a, The total 25 μL mixture from Hy3™-labeled samples with 25 μL hybridization buffer were first denatured for 2 min at 95°C, incubated on ice for 2 min b, Then hybridized to the microarray for 16–20 h at 56°C in a 12-Bay Hybridization Systems (Hybridization System - Nimblegen Systems, Inc., Madison, WI, USA) c, Following hybridization, the slides were achieved, washed several times using Wash buffer kit (Exiqon)
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| Scan protocol |
Then the slides were scanned using the Axon GenePix 4000B microarray scanner (Axon Instruments, Foster City, CA).
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| Description |
miRNA expression profiles in AML exosomes after GDC-0152 treatment.
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| Data processing |
Scanned images were then imported into GenePix Pro 6.0 software (Axon) for grid alignment and data extraction. Replicated miRNAs were averaged and miRNAs that intensities>=30 in all samples were chosen for calculating normalization factor. Expressed data were normalized using the Median normalization. After normalization, significant differentially expressed miRNAs between two groups were identified through Fold change and P-value. Differentially expressed miRNAs between two samples were filtered through Fold change. Finally, hierarchical clustering was performed to show distinguishable miRNA expression profiling among samples.
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| Submission date |
Dec 26, 2021 |
| Last update date |
Dec 27, 2021 |
| Contact name |
Xinyi Long |
| E-mail(s) |
2020121105@stu.cmu.edu.cn
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| Organization name |
China Medical university
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| Street address |
Shenyang
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| City |
Shenyang |
| State/province |
State... |
| ZIP/Postal code |
no |
| Country |
China |
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| Platform ID |
GPL17107 |
| Series (1) |
| GSE192628 |
Analysis of microRNA expression profiles in exosomes derived from acute myeloid leukemia by p62 knockdown and effect on angiogenesis |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM5753559_Cont_drug.gpr.gz |
979.5 Kb |
(ftp)(http) |
GPR |
| Processed data are available on Series record |
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