|
| Status |
Public on Jan 01, 2018 |
| Title |
NC-3 |
| Sample type |
RNA |
| |
|
| Source name |
KGN cell lines
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: KGN
treatment: treated with LV-negative control
|
| Treatment protocol |
We selected three target sequences to construct the lentiviral shRNAs (LV-PWRN2-homo-502, LV-PWRN2-homo-1574, LV-PWRN2-homo-1261) including the negative control (LV-NC). The target sequences were used to design two complementary oligonucleotides, which were synthesized and cloned into pGLV3/H1/GFP+Puro Vector (GenePharma, China). The positive purified lentiviral shRNA-expressing plasmids were transfected with the packaging plasmids into 293T cells for lentivirus generation (Genepharma, China). Then, the vectors described above were used to infect KGN cells. Stable KGN cell lines were selected using 3ug/mL bulk puromycin-resistance culturing (puromycin, Sigma, St Louis, MO, USA) for 5 days. After that, the cells were examined microscopically for lentiviral GFP expression and analyzed by real-time PCR.
|
| Growth protocol |
the KGN cells were cultured in 1:1 Dulbecco’s modified Eagle’s medium and Ham’s F-12 medium (DMEM/ F12; Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% foetal bovine serum (FBS) and antibiotics (100 IU/mL penicillin and 100 μg mL streptomycin). On the day before the Lentivirus transfection, the KGN cells were placed into the medium without serum and incubated overnight.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA isolation was performed using the Qiagen RNeasy Mini Kit (Qiagen, Hilder, Germany) according to the manufacturer’s instructions.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 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.
|
| |
|
| Hybridization protocol |
1.5 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 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) 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.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan.
|
| Description |
Gene expression of KGN cell lines treated with negative control lentivirus vector
|
| Data processing |
The lncRNA+mRNA array data were analyzed for data summarization, normalization and quality control by using the GeneSpring software V11.5 (Agilent). The data were normalized by percentile 75 normalization. To select the differentially expressed genes, we used threshold values of ≥2.0 and ≤-2.0 -fold change and a Benjamini-Hochberg corrected p value of 0.05.
|
| |
|
| Submission date |
Apr 13, 2017 |
| Last update date |
Jan 01, 2018 |
| Contact name |
Xin Huang |
| E-mail(s) |
huangxin@51mch.com
|
| Organization name |
Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine
|
| Department |
Reproductive Medicine Centre
|
| Street address |
No.2699 Gaoke east road
|
| City |
Shanghai |
| State/province |
Shanghai |
| ZIP/Postal code |
210000 |
| Country |
China |
| |
|
| Platform ID |
GPL20115 |
| Series (1) |
| GSE97772 |
Expression profiles of lncRNAs and mRNAs in KGN/shPWRN2 cell lines |
|
