Affymetrix GeneChip Human Genome HG-U133A [HG-U133A]

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In this study the generic impact of protein aggregation (aggregation of proteins not associated with neurodegenerative disease) on gene expression in cultured cells was investigated by DNA microarray technology. The survey of gene expression showed that the Hsp40, Hsp70 and Hsp105 genes, all of which have documented aggregation suppression activity, were up-regulated. Unexpectedly, the survey also showed increased expression of the MEK5 gene with concomitant silencing of the MEK3 gene. The expression pattern of MEK5 at the mRNA and protein levels aligns with the kinetics of aggregate formation and dissolution. Cell viability was unaffected by protein aggregates. These findings are of particular importance for chronic neurodegenerative diseases where the intraneuronal accumulation of aggregate-prone proteins are a major characteristic of the diseases. The identification of changes in MEK5 gene expression have been observed in Alzheimer-related diseases which provides new diagnostic and therapeutic avenues in these diseases. The molecular neuropathological findings would not have occurred without the generic microarray analyses.

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Transcription profiling of generic impact of protein aggregation (aggregation of proteins not associated with neurodegenerative disease) on gene expression in human cultured cells

In this study the generic impact of protein aggregation (aggregation of proteins not associated with neurodegenerative disease) on gene expression in cultured cells was investigated by DNA microarray technology. The survey of gene expression showed that the Hsp40, Hsp70 and Hsp105 genes, all of which have documented aggregation suppression activity, were up-regulated. Unexpectedly, the survey also showed increased expression of the MEK5 gene with concomitant silencing of the MEK3 gene. The expression pattern of MEK5 at the mRNA and protein levels aligns with the kinetics of aggregate formation and dissolution. Cell viability was unaffected by protein aggregates. These findings are of particular importance for chronic neurodegenerative diseases where the intraneuronal accumulation of aggregate-prone proteins are a major characteristic of the diseases. The identification of changes in MEK5 gene expression have been observed in Alzheimer-related diseases which provides new diagnostic and therapeutic avenues in these diseases. The molecular neuropathological findings would not have occurred without the generic microarray analyses.


Affymetrix:Protocol:Hybridization-Unknown

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-MEXP-9622

Total RNA was extracted from six 100mm plates of cells expressing GFP and His-FLAG (control cells) and six plates of cells expressing GFP-S8 ATPase and FLAG-mGluR1ƒÑ (test cells) at each time point. Six millilitres of TRIzolTM reagent (Life Technologies) was added to each plate and the RNA was extracted using the manufacturer¡¦s protocol. RNA samples from control cells or test cells were pooled and resuspended in RNAse-free water and quantified spectrophotometrically. The integrity of all samples was confirmed by gel electrophoresis, demonstrating a 2-fold excess of 28S to 18S RNA, with no evidence of degradation. RNA Samples were then subjected to further purification with Qiagen RNeasy minikit and measured spectophotometrically to ensure that only those samples with A260/280= 1.9.2.0 were used for subsequent cRNA generation and microarray hybridization.  

P-MEXP-9623

Samples of RNA (13ug) were used for cRNA preparation. The first and second strand cDNA synthesis was performed using the SuperScript Choice System (Life Technologies) according to the manufacturer¡¦s instructions except using an oligo(dT) primer containing a T7 RNA polymerase promoter site. Labelled cRNA was prepared using the BioArray„· RNA Transcript labelling kit (Enzo Life Sciences, Farmingdale, NY, USA) in total reaction volumes of 40ƒÝl. Biotin-labelled CTP and UTP (Enzo) were used in the reaction together with unlabelled NTPs. Following in vitro transcription, the unincorporated nucleotides were removed using RNeasy columns (Qiagen). Fifteen micrograms of cRNA were fragmented at 94¢XC for 35 min in a fragmentation buffer containing 40 mM Tris-acetate pH 8.1, 100 mM KOAc, 30 mM MgOAc. Prior to hybridization, the fragmented cRNA in a 6x SSPE-T hybridization buffer (1 M NaCl, 10 mM Tris pH 7.6, 0.005% Triton X-100) was heated to 95¢XC for 5 min and subsequently to 40¢XC for 5 min, before loading onto the Affymetrix probe array cartridge.

P-MEXP-9624

Fifteen micrograms of cRNA were fragmented at 94°C for 35 min in a fragmentation buffer containing 40 mM Tris-acetate pH 8.1, 100 mM KOAc, 30 mM MgOAc. Prior to hybridization, the fragmented cRNA in a 6x SSPE-T hybridization buffer (1 M NaCl, 10 mM Tris pH 7.6, 0.005% Triton X-100) was heated to 95°C for 5 min and subsequently to 40°C for 5 min, before loading onto the Affymetrix probe array cartridge. The probe array was then incubated for 16 h at 40°C with constant rotation (60 r.p.m.). Washing and staining procedures were  performed in the Affymetrix Fluidics Station. The probe array was exposed to 10 washes in 6x SSPE-T at 25°C followed by four washes in 0.5x SSPE-T at 50°C. The biotinylated cRNA was stained with a streptavidin–phycoerythrin conjugate, 10 mg/ml (Molecular Probes, Eugene, OR), in 6x SSPE-T for 30 min at 25°C followed by 10 washes in 6x SSPE-T at 25°C. The probe arrays were scanned at 560 nm using a confocal laser-scanning microscope with an argon ion laser as the excitation source (Hewlett Packard GeneArray Scanner G2500A). The readings from quantitative scanning were analysed by the Expressionist Analyst software (version 5.0.3) from Genedata AG (Basel, Switzerland), which was also used for normalization of the different arrays prior to the analysis.

P-MEXP-9656

Sample extracted 0 hrs after transfection incubation period

P-MEXP-9657

Sample extracted 2 hrs after transfection incubation period

P-MEXP-9658

Sample extracted 4 hrs after transfection incubation period

P-MEXP-9659

Sample extracted 8 hrs after transfection incubation period

P-MEXP-9660

Sample extracted 16 hrs after transfection incubation period

P-MEXP-9661

Sample extracted 24 hrs after transfection incubation period

P-MEXP-9662

Sample extracted 48 hrs after transfection incubation period

P-MEXP-9663

Sample extracted 72 hrs after transfection incubation period

P-MEXP-9751

HEK293 cells were co-transfected with empty vectors by the calcium phosphate method. Culture plates (100mm) at 50-70% confluence were selected and media changed 3 hrs prior to transfection. To prepare the initial CaCl2-DNA solution for each 100mm plate, sterile deionised H2O, 10ƒÝg of purified DNA (each vector) and 83ƒÝl of 1.5M CaCl2 were added sequentially. The CaCl2-DNA solutions were made up to 500ƒÝl with sterile water. An aliquot of 500ƒÝl of 2x HBS (HEPES-Buffered-Saline; 50mM HEPES, 280mM NaCl, 1.5mM Na2HPO4, pH 7.1) was also prepared for each 100mm transfection plate. The prepared solutions of CaCl2-DNA and 2x HBS were then slowly mixed in a dropwise fashion with concomitant, vigorous shaking (or slow vortexing). The resulting Ca„ªPO4-DNA solution (1ml), was left to incubate for 15-20 mins at room temperature. Cell pellets derived from each trypsinised confluent plate were re-suspended in 3ml of medium (DMEM/10%FBS) and mixed thoroughly with 1ml of the Ca„ªPO4-DNA solution. The final mixture was made up to 10ml with further addition of medium and was split evenly into 2 plates. All plates containing transfected cells were finally placed in a 5% CO2 (37¢XC) incubator and harvested at the times indictated in the experimental design.

P-MEXP-9753

HEK293 cells were co-transfected with vectors expressing S8 ATPase/mGluR1£\ by the calcium phosphate method. Culture plates (100mm) at 50-70% confluence were selected and media changed 3 hrs prior to transfection. To prepare the initial CaCl2-DNA solution for each 100mm plate, sterile deionised H2O, 10ƒÝg of purified DNA (each vector) and 83ƒÝl of 1.5M CaCl2 were added sequentially. The CaCl2-DNA solutions were made up to 500ƒÝl with sterile water. An aliquot of 500ƒÝl of 2x HBS (HEPES-Buffered-Saline; 50mM HEPES, 280mM NaCl, 1.5mM Na2HPO4, pH 7.1) was also prepared for each 100mm transfection plate. The prepared solutions of CaCl2-DNA and 2x HBS were then slowly mixed in a dropwise fashion with concomitant, vigorous shaking (or slow vortexing). The resulting Ca„ªPO4-DNA solution (1ml), was left to incubate for 15-20 mins at room temperature. Cell pellets derived from each trypsinised confluent plate were re-suspended in 3ml of medium (DMEM/10%FBS) and mixed thoroughly with 1ml of the Ca„ªPO4-DNA solution. The final mixture was made up to 10ml with further addition of medium and was split evenly into 2 plates. All plates containing transfected cells were finally placed in a 5% CO2 (37¢XC) incubator and harvested at the times indictated in the experimental design.

Transcription profiling of generic impact of protein aggregation (aggregation of proteins not associated with neurodegenerative disease) on gene expression in human cultured cells

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