|
| Status |
Public on Jun 01, 2019 |
| Title |
Hela_6h_SO_sample2 |
| Sample type |
RNA |
| |
|
| Source name |
Hela_6h_SO
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: cervical cancer
cell line: Hela
treatment: infected with sense oligonucleotides (SO)
sample type: control
gender: female
time post transfection: 6h
|
| Treatment protocol |
For transfection procedure Thermo Fisher Scientific Protocol for Oligofectamine reagent was used. We transfected cell with antisense oligonucleotides (ASO) specifically inducing CSB mRNA degradation . Moreover, we used three different experimental controls: mock treated cells (CTRL), transfection reagents alone (OLF) and cells treated with sense oligonucleotides (SO), the reverse of ASO. Briefly the day before transfection 1x10^5 cells were plated in 6-well dishes using medium without antibiotics. Immediately before transfection the medium was replaced with Optimem and oligonucleotides (200 nM final concentration) were delivered using Oligofectamine transfection reagent. The cells were transfected with ASO or SO or simply exposed to the transfectant reagent (OLF). Four hours after transfection Optimem was replaced with fresh complete medium.
|
| Growth protocol |
HeLa tumor cell lines were grown in DMEM containing 10% FCS and Gentamicin in a 5% CO2 humidified atmosphere at 37 Celsius degrees.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted at 2 or 8h after medium adding. Total RNA was isolated from HeLa cells using Absolutely RNA Miniprep kit (Agilent Technologies) for microarray analysis and qRT-PCR. Total RNA was extracted using Trizol (Invitrogen) and DNAse treated by Qiagen columns. Quality and integrity of each sample was checked using the Agilent BioAnalyzer 2100 (Agilent RNA 6000 nano kit): samples with a RNA Integrity Number (RIN) index lower than 8.0 were discarded. All the experimental steps involving the labelling, hybridization and washing of the samples were done following the one-color Agilent protocol.
|
| Label |
Cy3
|
| Label protocol |
For each sample 200ng of Poly A+RNA were labelled, using Cyanine 3-CTP (Agilent) in separate labelling reactions, with the Agilent Low Imput Linear Amplification Kit and purified with Qiagen’s RNeasy mini spin columns.
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| |
|
| Hybridization protocol |
One-color mixes of labelled cRNA pairs were hybridized to Agilent 8X60K whole human genome oligonucleotide microarrays (Grid ID 039494) , at 65°C for 17 hours using Agilent’s Gene Expression Hybridization Kit. The hybridized microarrays were disassembled at room temperature in Agilent Gene Expression Wash Buffer 1. After the disassembly, the microarrays were washed in Gene Expression Buffer 1 for one minute at room temperature, followed by washing with Gene Expression Wash Buffer 2 for one minute at 37°C. The microarrays were then treated with acetonitrile for one minute at room temperature.
|
| Scan protocol |
Post-hybridization image acquisition was accomplished using the Agilent Scanner G2564C. Data extraction from the 20 bits TIFF images was accomplished by Agilent Feature Extraction ver 10.7 software using the standard Agilent one-color gene expression extraction protocol (GE1_107_Sep09).
|
| Description |
Standard commercial immotalized cell line originally derived from cervical tumor
|
| Data processing |
The “gProcessedSignal” data column of the Feature Extraction *.txt output file was used, containing the median signals corrected by spatial and multiplicative detrend. Data quality filtering was performed using Microsoft Excel, discarding features with the Feature Extraction flag gIsWellAboveBG=0 in any sample, wich is a statistical method approximately equivalent to discarding features with a Signal/Noise ratio smaller than 2.0-3.0, where Signal = (median of the spot - median spot background level) and Noise is the Standard Deviation of the median spot background. Data were normalized to the 75th percentile in Log2 scale. Data were were further filtered by excluding mRNA probes with a coefficient of variation |Dev.st/Mean |>5% in Log2 expression values between biological replicates, across all samples, to obtain a final selection 17703 transcripts. Differentially expressed genes were selected by a combination of fold change and moderated T-test thresholds by the R-Bioconductor tool Limma (p-value<0.05; |Log2 fold-change ratio| >0.56 equivalent to 1.5 fold in linear scale).
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| |
|
| Submission date |
Jun 14, 2016 |
| Last update date |
Jun 01, 2019 |
| Contact name |
Ivan Arisi |
| E-mail(s) |
[email protected]
|
| Phone |
+39-06-49255230
|
| Organization name |
European Brain Research Institute
|
| Department |
Bioinformatics Facility
|
| Street address |
viale Regina Elena 295
|
| City |
Roma |
| ZIP/Postal code |
00161 |
| Country |
Italy |
| |
|
| Platform ID |
GPL17077 |
| Series (1) |
| GSE83367 |
CSB ablation induced apoptosis is mediated by Increased Endoplasmic Reticulum Stress Response: a gene expression study |
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