|
| Status |
Public on Jul 31, 2012 |
| Title |
Microcystin-LR 25MC10 |
| Sample type |
RNA |
| |
|
| Source name |
WRL-68 treated with MC-LR
|
| Organism |
Homo sapiens |
| Characteristics |
cell name: WRL-68
treatment: Microcystin-LR
|
| Treatment protocol |
WRL-68 cells were seeded on 25 cm2 flasks at a density of 2×104 cells/mL (2×105 cells per flask) and exposed to 10 µg/L MC-LR (final concentration) and incubated. After 72 h, the cells were then passaged and exposed to another 10 µg/L MC-LR. The same treatment with every passage for 25 passages and the cells were labeled 25MC10. The control cells were simultaneously treated with phosphate-buffered saline (PBS). All treatments were performed in triplicate.
|
| Growth protocol |
Human cell line WRL-68 (CL-48 from ATCC, Manassas, VA, USA) was grown in 25 cm2 flasks with Dulbecco’s modified Eagle’s minimal essential medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated certified fetal calf serum (FCS; Sijiqing Biological Engineering Materials Co., Ltd., Hangzhou, Zhejiang, PR China), 100 U/mL penicillin, and 100 mg/mL streptomycin at 37 °C in a humidified chamber with 5% CO2. The cells were passaged at the estimated log growth phase until a sufficient number of 80%–90% confluent 25 cm2 flasks were obtained for each experiment.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from the control cells (n=3) and that from the 25MC10 cells (n=3) were isolated using TRIzol reagent (Invitrogen) following the manufacturer’s instructions.
|
| Label |
Cy3
|
| Label protocol |
WRL-68 cells were seeded on 25 cm2 flasks at a density of 2×104 cells/mL and incubated for 72 h. The cells were then passaged and exposed to 10 µg/L MC-LR (final concentration). The cells were labeled 25MC10, following the passage number and the final concentration of MC-LR used. The control cells were simultaneously treated with phosphate-buffered saline (PBS). All treatments were performed in triplicate.
|
| |
|
| Hybridization protocol |
Samples were sent to Exiqon where they were labeled and hybridized into the miRCURY LNA microarray (miRbase 11.0 human array)
|
| Scan protocol |
The processed slides were scanned by using an Axon 4000B microarray Scanner, with the laser set at 635 nm and Power 80% and the photomultiplier set at 70% with a scan resolution of 10 µm.
|
| Description |
WRL-68 cells were seeded on 25 cm2 flasks at a density of 2×104 cells/mL (2×105 cells per flask) and exposed to 10 µg/L MC-LR (final concentration) and incubated. After 72 h, the cells were then passaged and exposed to another 10 µg/L MC-LR. The same treatment with every passage for 25 passages and the cells were labeled 25MC10.
|
| Data processing |
The intensity of green signal is calculated after background subtraction and replicated spots on the same slide have been averaged by getting a median intensity. 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. The profiling identified a subset of the total number of miRNAs analyzed by the miRCURY™ array that are differentially expressed. Unsupervised hierarchical clustering was performed on miRNA expression profiling. We also show the scatter plot comes with Correlation R-value in the Correlation & Scatter Plot sheet in miRNA_Data_Analysis_Report.
|
| |
|
| Submission date |
Jul 11, 2011 |
| Last update date |
Jul 31, 2012 |
| Contact name |
Lizhi Xu |
| E-mail(s) |
xulizhi@nju.edu.cn
|
| Phone |
+086-83686495
|
| Organization name |
Nanjing University
|
| Department |
School of Medicine
|
| Street address |
22 Hankou Road
|
| City |
Nanjing |
| State/province |
Jiangsu |
| ZIP/Postal code |
210093 |
| Country |
China |
| |
|
| Platform ID |
GPL7723 |
| Series (1) |
| GSE30560 |
Alteration of MicroRNA Expression Profile Linked to Microcystin-LR-Induced Tumorigenicity in Human WRL-68 Cell Line |
|
