|
| Status |
Public on Jul 14, 2014 |
| Title |
H5N1-NS1-Transfected Embryonic kidney (293T) Cells Replicate 1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Control Embyronic kidney(293T) Cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: 293T
passage: 60
cell type: embryonic cells derived from kidney
|
| Growth protocol |
293T cells were grown in modified Eagle’s medium (DMEM; Invitrogen Life Technologies, Carlsbad, CA,USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen Life Technologies) serum,100 units/ml penicillin, 100 ug/ml streptomycin in tissue culture flasks (Corning, USA) at 37°C in a CO2 incubator.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Cy3
|
| Label protocol |
1 µg of total RNA were primed with 1 µl of 100 µM T7 Oligo(dT) primer at 70°C for 10 min, then reversed transcribed at 42°C for 2 h in the presence of ArrayScript reverse transcriptase (Aminoallyl MessageAmp II aRNA amplification kit[Ambion]), the first strand cDNA was used for the synthesis of second strand using poly-A T7 primer and DNA polymerase using manufacturer's instruction (Ambion). The purified cDNA was used for in-vitro transcription to synthesize amino-allyl-Modified aRNA using 5-(3-amino allyl)-UTP in a ratio of 1:1 along with unmodified UTPs(25mM) and 25mM of ATP, CTP and GTP.The purified aRNA was used for Dye Coupling reaction using Cy-dyes. The purified dye labelled aRNA was further used for hybridization procedure.
|
| |
|
| Channel 2 |
| Source name |
H5N1-NS1-Transfected Embryonic kidney (293T) Cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: 293T transfected with pcDNA 3.0(-)containing H5N1-NS1 gene
passage: 60
cell type: embryonic cells derived from kidney
|
| Treatment protocol |
PCR amplification and standard cloning techniques were used to insert NS1 gene in plasmid pCDNA 3.1-(Invitrogen). The plasmid with insert was transfected into 293T cells using TransIT-LT1 (Mirus biosciences) transfection reagent.
|
| Growth protocol |
293T cells were grown in modified Eagle’s medium (DMEM; Invitrogen Life Technologies, Carlsbad, CA,USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen Life Technologies) serum,100 units/ml penicillin, 100 ug/ml streptomycin in tissue culture flasks (Corning, USA) at 37°C in a CO2 incubator.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Cy5
|
| Label protocol |
1 µg of total RNA were primed with 1 µl of 100 µM T7 Oligo(dT) primer at 70°C for 10 min, then reversed transcribed at 42°C for 2 h in the presence of ArrayScript reverse transcriptase (Aminoallyl MessageAmp II aRNA amplification kit[Ambion]), the first strand cDNA was used for the synthesis of second strand using poly-A T7 primer and DNA polymerase using manufacturer's instruction (Ambion). The purified cDNA was used for in-vitro transcription to synthesize amino-allyl-Modified aRNA using 5-(3-amino allyl)-UTP in a ratio of 1:1 along with unmodified UTPs(25mM) and 25mM of ATP, CTP and GTP.The purified aRNA was used for Dye Coupling reaction using Cy-dyes. The purified dye labelled aRNA was further used for hybridization procedure.
|
| |
|
| |
| Hybridization protocol |
Control and Experimental purified labelled aRNA were mixed in equal concentrations in terms of dye labelling and the mixture was dried completely. To the dired mixture 140µl of Hybridization buffer (Pronto Hybridization kit, Corning) was added and denatured at 95˚C for 5 minutes. The denatured mixture was allowed to hybridize onto a pre-soak, pre-hybridized(reagents from Pronto kit[Corning] microarray slide.
|
| Scan protocol |
Scanned on Scan array (Perkin Elmer) scanner.
Images were quantified using Scan array express Software (version 1.5).
|
| Data processing |
LOWESS normalized, background subtracted data obtained from log2 of processed Red signal/processed Green signal.
|
| |
|
| Submission date |
Jul 06, 2012 |
| Last update date |
Jul 14, 2014 |
| Contact name |
Sanjay Mukherjee |
| Organization name |
University of Pune
|
| Department |
Department of Zoology
|
| Lab |
Molecular Biology research Lab.
|
| Street address |
Ganeshkhind
|
| City |
Pune |
| State/province |
Maharashtra |
| ZIP/Postal code |
411007 |
| Country |
India |
| |
|
| Platform ID |
GPL15774 |
| Series (1) |
| GSE39155 |
Human Embryonic Kidney(293T) Cells: Control vs. Transfected |
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