Affymetrix GeneChip Murine 11K Genome [Mu11KsubA]

Affymetrix GeneChip Murine 11K Genome [Mu11KsubB]

The protein kinase PKR: a molecular clock that sequentially activates survival and death programs.

rawData

scan

processedDataMatrix

PKR is an interferon induced serine/threonine protein kinase, that is activated by double stranded RNA. PKR plays an important role in the antiviral defense by interferon. In addition to its role in translation, PKR participates in several signaling pathways to transcription. The goal of this experiment is to study the role of PKR in regulating gene expression in our NIH 3T3 inducible cell line, which could overexpress PKR wt protein after the removal of tetracycline (Donze O, Dostie J, Sonenberg N. (1999) Virology 256: 322-9).

Transcription profiling of human NIH 3T3 inducible cell line to study the role of PKR in regulating gene expression

PKR is an interferon induced serine/threonine protein kinase, that is activated by double stranded RNA. PKR plays an important role in the antiviral defense by interferon. In addition to its role in translation, PKR participates in several signaling pathways to transcription. The goal of this experiment is to study the role of PKR in regulating gene expression in our NIH 3T3 inducible cell line, which could overexpress PKR wt protein after the removal of tetracycline (Donze O, Dostie J, Sonenberg N. (1999) Virology 256: 322-9).


Affymetrix:Protocol:ExpressionStat

Title: Affymetrix CHP Analysis (ExpressionStat). Description:

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-MEXP-2199

NIH3T3-PKR cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal calf serum (FCS) in the presence of tetracycline (1 µg/ml). To induce expression of PKR, cells were washed twice with Tris-buffered saline to remove tetracycline and fed with fresh DMEM containing 5% FCS.

P-MEXP-2200

The cells were harvested by removing the culture media and adding Trizol reagent (Gibco BRL Life Technologies) directly to the culture dish. Total cellular RNA was then prepared according to the manufacturer’s instructions, quantified by spectrophotometry and analyzed by gel electrophoresis.

P-MEXP-2201

P-MEXP-2203

The hybridization mixture was prepared by mixing 15 micrograms of biotinylated probe with Control Oligonucleotide B2 (final concentration 50 pM) (Affymetrix), Herring Sperm DNA (final concentration 0.1 mg/ml) (Research Genetics), Acetylated BSA (final concentration 0.5 mg/ml) (Gibco BRL Life Technologies) in a final volume of 300 microlitres of 1X MES Hybridization Buffer (100 mM MES, 1 M NaCl, 20 mM EDTA, 0.01% Tween-20) (all reagents from Sigma). The hybridization mix was denatured for 10 minutes at 99C, incubated for 5 minutes at 45C and spun for 5 minutes in a benchtop microfuge. The microarray was warmed to room temperature and prehybridized in 1X hybridization buffer for 10-20 minutes at 45C. The prehybridization solution was removed and 150 microlitres of the hybridization mix was added to the array. The array and probe fragments were incubated at 45C overnight (16-20 hours). Following hybridization, non-specifically bound probe was removed by washing using the GeneChip Fluidics Station 400 (Affymetrix). In total, ten low stringency washes (6X SSPE, 0.01% Tween-20, 0.005% Antifoam) and four high stringency washes (100 mM MES, 0.1 M NaCl, 0.01% Tween-20, 50C) were performed (all reagents, Sigma).

P-MEXP-2253

Control sample (PKR wt 5-) incubated in Tetracycline-containing medium.

P-MEXP-2254

Untreated sample (PKR wt 5+) incubated in Tetracycline-free medium.

Transcription profiling of human NIH 3T3 inducible cell line to study the role of PKR in regulating gene expression

Submitter Supplied Information

Samples