Kingfisher duo rna extraction machine

Total RNA was extracted from ~20 mg of six snap-frozen GBM tissue from the same cohort of patients included in DAB IHC analysis, using the MagJET RNA kit (cat# k2731, Thermo Scientific) and the Kingfisher Duo RNA extraction machine (Thermo Scientific). All samples were quantitated and quality controlled with the NanoDrop 2000 Spectrophotometer (Thermo Scientific) and the Qubit 2.0 Fluormeter (Thermo Scientific). The samples with A260/A230 ≥1.5 and A260/A280 ~2 were used for further analysis. The integrity of the RNA was assessed by New Zealand Genomics Ltd. (Dunedin, NZ) using Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA). The isolated RNA was then subjected to NanoString nCounter™ Gene Expression Assay (NanoString Technologies, Seattle, WA, USA) as completed by New Zealand Genomics Ltd. (Dunedin, New Zealand), according to the manufacturer’s protocol. Probes for the genes encoding NANOG (XM_011520850.1), SOX2 (NM_003106.3), SALL4 (NM_020436.3), OCT4 (NM_001159542.1), and STAT3 (NM_139276.2) and the housekeeping gene GAPDH (NM_002046.3) were designed and synthesized by NanoString Technologies. Raw data were analyzed using nSolver™ software (NanoString Technologies) using standard settings and was normalized against the housekeeping gene.

Total RNA was extracted from ~20 mg of snap-frozen GBM tissue (n = 6) from the same cohort of patients included in DAB IHC staining using the MagJET RNA kit (cat# k2731, Thermo Scientific) and the Kingfisher Duo RNA extraction machine (Thermo Scientific). All samples were quantitated and quality controlled with the NanoDrop 2000 Spectrophotometer (Thermo Scientific) and the Qubit 2.0 Fluorimeter (Thermo Scientific). The samples with A260/A230 ≥ 1.5 and A260/A280 ~ 2 were used for further analyses. The integrity of the RNA was assessed by the New Zealand Genomics Ltd. (Dunedin, New Zealand) using Agilent 2100 BioAnalyzer (Agilent Technologies). The isolated RNA was then subjected to NanoString nCounter™ Gene Expression Assay (NanoString Technologies, Seattle, WA, USA) as completed by New Zealand Genomics Ltd (Dunedin, New Zealand), according to the manufacturer’s protocol. Probes for the genes encoding the PRR (NM_005765.2), ATIIR1 (NM_000685.3), ATIIR2 (NM_000686.3), ACE (NM_000789.2) and the housekeeping gene, and GAPDH (NM_002046.3) were designed and synthesized by NanoString Technologies. Raw data were analyzed with Microsoft Excel using standard settings and were normalized against the housekeeping genes.