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| Status |
Public on Aug 04, 2016 |
| Title |
cell_24h_pEGFP-N1-miR3131-WT vetor_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Sk-Hep-1 cell line, transfected pEGFP-N1-miR3131-WT vetor 24h, replicate 1
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| Organism |
Homo sapiens |
| Characteristics |
cell line: Sk-Hep-1, Hepatocellular carcinoma cell line
transfection vector: pEGFP-N1-miR3131-WT
|
| Treatment protocol |
The fragment of 366bp including the insert allele of rs57408770 in pre-miR-3131 was directly synthesized by Genewiz Company (Suzhou, China) and cloned into BamHI and EcoRI sites of pEGFP-N1 (Cat#6085-1, Promega) yielding the wild-type vector (pEGFP-N1-miR3131-WT). The mutant-type vector (pEGFP-N1-miR3131-MT) including the delete allele of rs57408770 was generated using QuikChange Lightening Site-Directed Mutagenesis Kit (Cat # 210518, Stratagene). The resulting constructs were verified by direct sequencing. For overexpression experiments, cells were cultured in 6-well plates at a density of 200 000 cells per well the day before transfection. 2.5μg plasmids of empty vector pEGFP-N1, pEGFP-N1-miR3131-WT or pEGFP-N1-miR3131-MT were transfected into the growing cells (about 80%-90% confluent) using Lipofectamine2000 (Invitrogen). After 24 hours of the transfection, the cells were collected for subsequent experiments.
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| Growth protocol |
Sk-Hep-1 cell line was cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in a humidified chamber supplemented with 5% CO2. Upon resuscitation, the cell lines were characterized by Genetic Testing Biotechnology Corporation (Suzhou, China) using short tandem repeat (STR) markers.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from cell line according to the manufacturer's protocol (Cat #74106, Qiagen). Total RNA was quantified by the NanoDrop ND-2000 (Thermo Scientific) and the RNA integrity was assessed using Agilent Bioanalyzer 2100 (Agilent Technologies).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.2 ug RNA using the One-Color LowInput Quick-Amp Labeling Kit (Cat # 5190-2305, Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (Cat #74106, QIAGEN). Dye incorporation and cRNA yield were checked with the NanoDrop ND-2000 Spectrophotometer.
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| |
|
| Hybridization protocol |
0.6 ug of Cy3-labelled cRNA (specific activity >9.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25ul containing 25x Agilent fragmentation buffer and 10x Agilent blocking agent following the manufacturer instructions. On completion of the fragmentation reaction, 25ul of 2xGEx Hybridization Buffer HI-RPM was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays 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 (G2505C) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to 100%).
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| Description |
After pEGFP-N1-miR3131-WT being transfected in sk-Hep-1 cells for 24h, human genome-wide gene expression profile chip was adopted to screen the differentially expressed genes.
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| Data processing |
Feature Extraction software (version10.7.1.1, Agilent Technologies) was used to analyze array images to get raw data. Genespring (version 12.5, Agilent Technologies) were employed to finish the basic analysis with the raw data. To begin with, the raw data was normalized with the quantile algorithm. The probes that at least 100% of the values in any 1 out of all conditions have flags in Detected were chosen for further data analysis. Differentially expressed genes were then identified through fold change. The threshold set for up- and down-regulated genes was a fold change>= 2.0. Afterwards, GO analysis and KEGG analysis were applied to determine the roles of these differentially expressed mRNAs.
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| |
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| Submission date |
Aug 03, 2016 |
| Last update date |
Aug 04, 2016 |
| Contact name |
wang chaoqun |
| E-mail(s) |
wangchaoqun880924@126.com
|
| Organization name |
Medical College of Soochow University
|
| Department |
Department of Forensic Medicine
|
| Street address |
Room 806-1, Engineering Building, North Campus ,Soochow University, No. 178, Ganjiang East Road, Gusu District
|
| City |
Suzhou |
| State/province |
Jiangsu |
| ZIP/Postal code |
215123 |
| Country |
China |
| |
|
| Platform ID |
GPL17077 |
| Series (1) |
| GSE85123 |
An Indel Polymorphism within pre-miR3131 Confers Risk for Hepatocellular Carcinoma |
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