Genechip 3 ivt express kit user manual

Total RNA was extracted from NIH3T3 fibroblasts treated with 15 μg ml^-1 purified p37 protein for 24 hours or non-treated NIH3T3 fibroblasts using the RNeasy® Mini Kit (Qiagen; Cat# 74104). Three biological replicates were taken of each treatment. Genomic contamination was screened by PCR and eliminated using Deoxyribonuclease I, Amplification Grade (Invitrogen; Cat#18068–015) per the manufacturer’s instruction. RNA integrity and quality was checked using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, California, USA).
RNA was amplified and cDNA synthesized according to instructions in the Genechip® 3’ IVT Express Kit User Manual (Affymetrix; Cat# P/N702646 Rev.8). Following biotin labelling of cDNA and fragmentation, samples were hybridized to Affymetrix Mouse Genome 430 2.0 Arrays, washed using a Genechip® Fluidics Station and scanned using the Genechip® Scanner 3000. Microarray data was processed using the Affymetrix® Expression Console™ Software 1.2 (Affymetrix; Cat# P/N 702387 Rev. 2) and CLC Genomics Workbench 4.7 (CLC bio, http://www.clcbio.com; Vat#DK28305087).

The RNA was prepared from PBMC lysates by SeqWright DNA Technology Services (Houston, TX, USA). Using standard procedures, total RNA was isolated using the Qiagen RNeasy Plus Mini Kit (Qiagen, Valencia, CA, USA), RNA concentrations were determined using a Nanodrop ND-100 spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE, USA), and RNA quality was determined by Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
Gene expression profiling was performed using Affymetrix Human Genome U133 Plus 2.0 microarray (Affymetrix, Inc., Santa Clara, CA, USA). The expression of ~47,000 transcripts were analyzed by using 100 ng of RNA sample for double-stranded cDNA synthesis, in vitro transcription of cRNA with biotin labelling and hybridization of cRNA on the microarray. The assay was performed using the Affymetrix standard protocol GeneChip® 3’ IVT Express Kit User Manual (P/N 702646 Rev. 1) and the GeneChip® Expression Analysis Technical Manual With Specific Protocols for Using the GeneChip Hybridization, Wash, and Stain Kit (P/N 702232 Rev. 3).

Isolation of total RNA was done as described previously78 (link). Target preparation, hybridization to arrays, washing, staining and scanning were carried out per manufacturer’s instructions (GeneChip^® 3’ IVT Express Kit User Manual, 2008, Affymetrix). Affymetrix GeneChip Operating Software 1.2.1 was used for washing and scanning in Fluidics Station 450 (Affymetrix) and Scanner 3000 (Affymetrix), respectively. For data analysis, the probe intensity (.cel) files of all 18 chips were imported into ArrayAssist^® (Stratagene). Normalization of data was carried out by using the GC-RMA algorithm (Gene Chip Robust Multiarray Analysis) implemented in the software. The genes after ANOVA analysis showing change of at least two-fold at a p-value of <0.05 under drought conditions were identified as differentially expressed genes. Further, these genes were annotated as per TIGR version 7. Redundant probe-sets were removed and genes with unknown functions were manually annotated by sequence retrieval (NCBI, KOME and TIGR) and domain search using SMART and Pfam databases. Further information was derived from Gene Ontology (GO), InterPro, Uniprot and published data. The gene lists were then divided into functional groups based on those previously suggested25 (link)26 (link).
The GC-RMA normalized data have been submitted to GEO under accession number GSE41647.