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| Status |
Public on Sep 13, 2018 |
| Title |
total RNA from RBM10 siRNA treated sample 3 |
| Sample type |
RNA |
| |
|
| Source name |
HEK 293 cell line
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK 293
sirna knockdown: RBM10 siRNA
|
| Treatment protocol |
After reaching 60% of confluency, HEK 293 cells was transfected with siRNA oligos using Oligofectamine reagent and cells were harvested 48-hours post-transfection.
|
| Growth protocol |
HEK 293 cell line was obtained from American Type Culture Collection and maintained in DMEM with 10% FBS and penicillin/streptomycin (50 U/ml) at 37°C in 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from HEK 293 cells transfected with RBM10 specific or control siRNA oligonucleotides using the RNeasy Mini Kit (Qiagen, Cat.No. 74004 ) with DNAse treatment as per manufacturer’s protocol. The RNA sample was eluted in 25uL of Nuclease free water. The concentration and purity of the RNA extracted were evaluated using the Nanodrop Spectrophotometer (Thermo Scientific; 1000). The integrity of the extracted RNA were analysed on the Bioanalyzer (Agilent; 2100 expert). We considered RNA to be of good quality based on the 260/280 values (Nanodrop), rRNA 28S/18S ratios and RNA integrity number (RIN) (Bioanalyzer).The purity and integrity of RNA was checked using Nanodrop (Thermo Scientific) and Bioanalyzer (Agilent)
|
| Label |
Cy3
|
| Label protocol |
Samples for Gene expression were labeled using Agilent Quick-Amp labeling Kit (p/n5190-0442). 500ng each of total RNA were reverse transcribed at 40°C using oligo dT primer tagged to a T7 polymerase promoter and converted to double stranded cDNA. Synthesized double stranded cDNA were used as template for cRNA generation. cRNA was generated by in vitro transcription and the dye Cy3 CTP(Agilent) was incorporated during this step. The cDNA synthesis and in vitro transcription steps were carried out at 40°C. Labeled cRNA was cleaned up using Qiagen RNeasyMini kit columns (Qiagen, Cat No: 74106) and quality assessed for yields and specific activity using the Nanodrop ND-1000.
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| |
|
| Hybridization protocol |
600ng of labeled cRNA sample were fragmented at 60ºC and hybridized on to a Agilent designed Human Gene expression Microarray 8x60K (AMADID No: 39494) arrays. Fragmentation of labeled cRNA and hybridization were done using the Gene Expression Hybridization kit of (Agilent Technologies, In situ Hybridization kit, Part Number 5190-0404). Hybridization was carried out in Agilent’s Surehyb Chambers at 65º C for 16 hours.
|
| Scan protocol |
The hybridized slides were washed using Agilent Gene Expression wash buffers (Agilent Technologies, Part Number 5188-5327) and scanned using the Agilent Microarray Scanner (AgilentTechnologies, Part Number G2600D).
|
| Description |
gene expression after treating with RBM10 siRNA
|
| Data processing |
Data extraction from Images was done using Feature Extraction software Version 11.5 of Agilent. Images were quantified using Feature Extraction Software (Version-11.5 Agilent). Feature extracted raw data was analyzed using GeneSpring GX software from Agilent.
Normalization of the data was done in GeneSpring GX using the 75th percentile shift (Percentile shift normalization is a global normalization, where the locations of all the spot intensities in an array are adjusted. [This normalization takes each column in an experiment independently, and computes the percentile of the expression values for this array, across all spots (where n has a range from 0-100 and n=75 is the median). It subtracts this value from the expression value of each entity) and fold expression values were obtained with respect to Specific control Samples].
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| |
|
| Submission date |
May 14, 2018 |
| Last update date |
Sep 14, 2018 |
| Contact name |
Rakesh S Laishram |
| E-mail(s) |
[email protected]
|
| Organization name |
Rajiv Gandhi Centre for Biotechnology(RGCB)
|
| Department |
Cardiovascular and Diabetes Biology Group
|
| Lab |
Cardiovascular and Diabetes Biology Lab
|
| Street address |
Thycaud P O, Poojappura
|
| City |
Trivandrum |
| State/province |
Kerala |
| ZIP/Postal code |
695014 |
| Country |
India |
| |
|
| Platform ID |
GPL21061 |
| Series (1) |
| GSE114437 |
Microarray based gene expression profiling of RNA binding protein, RBM10 in HEK 293 cells |
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