Ivt express kit

Microarray analysis was performed after 28 days of cultivation using the Human Genome U133 Plus 2.0 Arrays (Affymetrix). Procedures for cDNA synthesis, labelling and hybridization were carried out according to the manufacturer's protocol (Affymetrix). In brief, 50 ng of total RNA was used for first‐strand cDNA synthesis with an HPLC‐purified T7‐(dT)24 primer. Synthesis of biotin‐labelled cRNA and clean up was carried out using the IVT Express Kit (Affymetrix). For hybridization, 15 µg of fragmented cRNA was incubated with the chip in 200 µL of hybridization solution in Hybridization Oven 640 (Affymetrix) at 45°C for 16 hours. GeneChips were then washed and stained using the Affymetrix Fluidics Station 450 according to the GeneChip Expression Analysis Technical Manual (Rev. 2). Microarrays were scanned with the Affymetrix GeneChip Scanner 7G, and the signals were processed using GCOS (v.1.4; Affymetrix).

Total RNA that contained both mRNA and ncRNA was isolated from patient samples using the RNeasy kit (Qiagen Inc., Germantown, MD, USA). The quality of the total RNA was confirmed using the Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) and the RNA6000 Nano assay (Agilent). For each sample, the 3′ in vitro translation (IVT) express kit (Affymetrix, Santa Clara, CA, USA) synthesized biotin-labeled RNA target from 100 ng of the total RNA sample. The 3′ IVT kit contains hybrid primers that bind polyA (polydT) as well as random hexamer primers that bind ncRNA sequence. The kit generates cDNA to both polyA (coding) and non-polyA (non-coding) RNAs. A hybridization cocktail that included 10 μg of target was created for each sample. Samples were hybridized to the Genechip Primeview Human Gene Expression probe array cartridge (Affymetrix). The PrimeView Human Gene Expression Array provides comprehensive coverage of the human genome in a cartridge array format. The array is comprised of >530,000 probes covering >36,000 transcripts and variants, which represent >20,000 genes. Arrays contain probes for >20,000 total mature miRNAs, snoRNAs, ncRNAs, and pre-miRNAs. Those probes that demonstrated a cutoff greater or less than 2-fold from normal PCs were further analyzed.