Three batches of 6 × 105 SP1 and non-SP (MP1) cells were flow cytometrically sorted as biological replicates and stored in RNA Later (Ambion Inc., Austin, TX, USA). Total RNA was isolated using Qiagen’s All Prep DNA/RNA Mini Kit (Cat. No. 80204), and Human Genome U133 Plus 2.0 Arraychip (Affymetrix, Santa Clara, CA, USA) was used following the manufacturer’s instructions. Biotin-labeled targets (cRNA) were prepared from 100 ng of total RNA (two SP1 and three MP1 samples) using modified MessageAmp™-based protocols (Ambion Inc.). The standard Affymetrix protocol was followed for labeled cRNA, array hybridization, and washing. The hybridization step was carried out in an Affymetrix Model 640 hybridization oven at 45°C for 16 h and stained on an Affymetrix FS450 Fluidics station. Affymetrix GeneChip Scanner 3000 7G was used to scan the arrays. Affymetrix Statistical Algorithm MAS 5.0 (GCOS v1.3) algorithm was used to generate a summary of the image signal data, detection cells with all arrays scaled to 500, and gene annotations on the array. Statistical differential analysis was performed using the one-way ANOVA model to test the null hypothesis followed by a two-sample t-test for every gene and multiplicity correction to control the false discovery rate (FDR) at 0.05.
Model 640
Manufactured by Thermo Fisher Scientific
The Model 640 is a laboratory spectrophotometer designed for accurate and reliable absorbance measurements. It features a wavelength range of 190-1100 nm and can be used for a variety of applications in analytical chemistry, biochemistry, and life science research.
Automatically generated - may contain errors
Lab products found in correlation
Human genome u133 plus 2.0 array chip, by Thermo Fisher Scientific (1 mentions)
Genechip scanner 3000 7g, by Thermo Fisher Scientific (1 mentions)
Fs 450 fluidics station, by Thermo Fisher Scientific (1 mentions)
Rnalater, by Thermo Fisher Scientific (1 mentions)
Allprep dna rna mini kit, by Qiagen (1 mentions)
Genechip human genome u133 hg u133 plus 2.0 array, by Thermo Fisher Scientific (1 mentions)
Statistical algorithm mas 5, by Thermo Fisher Scientific (1 mentions)
G7 scanner, by Thermo Fisher Scientific (1 mentions)
Fluidics station, by Thermo Fisher Scientific (1 mentions)
3 protocols using model 640
1
Transcriptome Analysis of SP1 and MP1 Cells
2
Microarray Analysis of Gene Expression
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Gene expression analysis was performed in triplicate (three sham, three nsEP exposed and three heat-shock treated) using the Affymetrix GeneChip® Human Genome U133 (HG-U133) plus 2.0 Array that contains 54,675 probe sets. Briefly, two micrograms of RNA were used for preparation of biotin-labeled targets (cRNA) using MessageAmp™-based protocols (Ambion, Inc., Austin, TX). Labeled cRNA was fragmented (0.5 μg/μL per reaction) and used for array hybridization and washing. The cRNA was mixed with a hybridization cocktail, heated to 99°C for 5 min and then incubated at 45°C for 5 min. Hybridization arrays were conducted for 16 h in an Affymetrix Model 640 hybridization oven (45°C, and 60 rpm). Arrays were washed and stained on an FS450 Fluidics station and were scanned on a GeneChip® Scanner 3000 7G. Image signal data, detection calls and annotations were generated for every gene using the Affymetrix Statistical Algorithm MAS 5.0 (GeneChip® Operating Software v1.3). A log2 transformation was conducted and a Student’s t-test was performed for comparison of the nsEP exposed samples to the two control groups (sham and heat-shocked). We conducted multiple testing correction—Benjamini and Hochberg—to determine the false discovery rate, and statistically significant genes were identified using Bonferroni correction procedures.
3
Microarray Hybridization and Scanning
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The hybridization procedure was carried out for 16 hours at 56°C using a hybridization oven (model 640, Affymetrix). Microarrays were then washed and stained in an Affymetrix Fluidics Station. A computer CEL file containing microarray images was produced by an Affymetrix G7 scanner.
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