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| Status |
Public on Apr 06, 2020 |
| Title |
exosome-M2-3 |
| Sample type |
RNA |
| |
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| Source name |
M2 induced from Thp-1
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: exosome
|
| Treatment protocol |
the M2 group Thp-1 cells was induced by PMA (50 ng/ml) and IL-4 (20 ng/ml) for 24h, Then the culture medium was change into RPMI1640 and culture for 24h. The Thp-1 group was untreated for 24h, then change into RPMI1640 and culture for 24h.In order to isolate exosomes, we centrifuge the supernatants for two times (1000g × 10min, 3000g × 30min to deplete the cell or fragments), and then add the total exosome isolation kit (Life technology) over night, and then centrifuge for 10000g×1h.Total RNA was extracted by mirVanaTM RNA Isolation Kit (Applied Biosystem p/n AM1556).
|
| Growth protocol |
Thp-1 cells were cultured in RPMI1640+10%FBS , P6 was collected and devided into Thp-1 group and M2 group.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted by Trizol (Life Technology, 15596-018) following the manufacturer's instructions
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.2 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer. instructions,then followed by RNAeasy column purification (QIAGEN, Valencia, CA).
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| |
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| Hybridization protocol |
0.6 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 22.5ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers' instructions. On completion of the fragmentation reaction, 22.5ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to OE Human lncRNA Microarray V4.0 (Agilent) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
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| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 4x180k array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).
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| Description |
Gene expression of exosome secreted by M2 macrophage induced from Thp-1 cells after 24h.
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.1.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities as the raw data. Raw data were normalized in quantile algorithm with Genespring 13.0(Agilent). Probe that at least 1 out of 2 samples flagged as Detected were maintained.
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| |
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| Submission date |
Apr 06, 2017 |
| Last update date |
Apr 06, 2020 |
| Contact name |
xiaoduan li |
| E-mail(s) |
944810829@qq.com
|
| Organization name |
Department of Gynecology, Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, Shanghai , China.
|
| Street address |
Changle road no. 536
|
| City |
Shanghai |
| ZIP/Postal code |
201204 |
| Country |
China |
| |
|
| Platform ID |
GPL18402 |
| Series (1) |
| GSE97467 |
Different expression of miRNA between the exosomes secreted by monocyte and M2 macrophage |
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