Genechiph wt terminal labeling kit

Total RNA was isolated from CD34+ cells using RNeasy Mini Kit (Qiagen). RNA was isolated from CD34+ cells of five GH-treated patients at baseline, and in 3rd and 6th month of treatment, and of five control subjects, and next was pooled to generate the final RNA sample representing a particular group of patients. Sense-strand cDNA generated from total RNA using an Ambion WT Expression Kit (Life Technologies, Paisley, UK) was fragmented and labeled using the GeneChipH WT Terminal Labeling Kit (Affymetrix, Santa Clara, CA, USA) and hybridized onto an Affymetrix WT Array Strip. Hybridization as well as subsequent fluidics and scanning steps were performed using an Affymetrix GeneAtlasTM system (Affymetrix). Differences in the expression of the chosen genes and Gene Ontology (GO) terms were analyzed in the R programming environment using Bioconductor packages.

Ouabain octahydrate (Cat #O3125) was purchased from Sigma Aldrich (USA) and was diluted to 10 mM in ultra-pure water and further used for cell treatment. Trypsin-EDTA, fetal bovine serum, penicillin-streptomycin, Hank’s Balanced Salt Solution, and trypan blue were purchased from Paneco (Russia). Neurobasal Medium, Supplement B-27, and 0.5 mM GlutaMax were purchased from Gibco (USA). MMLV RT Kit and TaqMan qPCR mix-HS were purchased from Evrogen (Russia). Primers were purchased from DNA-synthesis (Russia). GeneChip Rat Gene 2.0 ST Array; GeneChip WT PLUS Reagent Kit; GeneChipH WT Terminal Labeling Kit; and GeneChip Hybridization, Wash, and Stain Kit were purchased from Affymetrix (USA). Poly-L-ornithine, RIPA buffer, MgCl₂, trichloroacetic acid, KCl, reagents for SDS-PAGE, cocktails of protease and phosphatase inhibitors, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and dimethyl sulfoxide were purchased from Sigma Aldrich. DC Protein Assay Kit was purchased from Bio-Rad (USA). Antibodies phospho-CREB (Ser133) (87G3) Rabbit mAb; β-Actin (D6A8) Rabbit mAb; Bcl-2 Rabbit pAb; Bax Rabbit pAb; and anti-rabbit IgG-HRP were purchased from Cell Signaling Technology (USA). SuperSignal West Pico Chemiluminescent Substrate, SuperSignal West Femto Chemiluminescent Substrate, DNase I, and PureLink RNA Mini Kit, SYTO13 were purchased from Thermo Fisher Scientific (USA).

Total RNA was isolated from CD34⁺ HPCs using RNeasy Mini Kit (Qiagen, USA). RNA was isolated from CD34⁺ cells of five GH-treated patients, the same at baseline, and in 3rd and 6th months of treatment, and of five control subjects, and was pooled to generate the final RNA sample representing a particular group in subsequent experimental procedures. Sense-strand cDNA generated from total RNA using an Ambion WT Expression Kit (Life Technologies, UK) was fragmented and labeled using the GeneChipH WT Terminal Labeling Kit (Affymetrix, USA) and hybridized onto an Affymetrix WT Array Strip. Hybridization as well as subsequent fluidics and scanning steps were performed using an Affymetrix GeneAtlasTM system (Affymetrix). Differences in the expression of the chosen genes and Gene Ontology (GO) terms were analyzed in the R programming environment using Bioconductor packages.