Allprep rna isolation kit

Four IDH1^wt and four IDH1^mut patient-derived GSC lines were cultivated as described (Table 1) [54 (link)]. In brief, cells were grown as floating neurospheres in DMEM/F-12 medium containing 20% BIT serum-free supplement, basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) at 20 ng/mL each (all Provitro, Berlin, Germany). To study expression changes, IDH1^mut and IDH1^wt GSCs were grown under normoxic (~20% O₂) and hypoxic conditions (1.5% O₂) as previously reported [24 (link),57 (link)]. Hypoxic conditions (1.5% O₂, 5% CO₂, 93.5% N₂) were established in an O₂- and CO₂-controlled tissue culture incubator (Nunc, Langenselbold, Germany) as previously described [58 (link)]. mRNA was harvested after 72 h using the Qiagen Allprep RNA isolation kit according to the manufacturer’s instructions.
The expression of several stem cell markers including CD133, SOX2, CD44, CSPG4, CD90, and nestin in the IDH1^mut GSCs has been shown by Kohanbash et al. [59 (link)]. Also for IDH1^wt GSCs elevated clonogeneity as well as expression of certain stem cell markers has been shown in previous publications from our laboratory [19 (link),60 (link)].

Two flasks of HUVECs from same passage were cultured with NG-ECGM and HG-ECGM for 72 h, respectively. Subsequently, HUVECs were harvested and transferred into RNAlater^TM RNA Stabilization Reagent (Qiagen, Hilden, Germany) for homogenization. MiRNAs were isolated using the Allprep RNA isolation kit (Qiagen) according to the manufacturer’s protocol. The RNA integrity number (RIN) was determined using the Agilent Bioanalyzer 2100 Expert (B.02.08.SI648, Agilent, Santa Clara, CA, USA). The RNA samples of HG and NG with RINs ranging from 7 to 10 were sent to the Beijing Genomics Institute (BGI, Shenzhen, China) to perform the miRNA sequencing. Filtered miRNAs were quantified by realigning reads to predicted miRNAs in QuickMIRSeq [15 (link)]. To decrease false positives, the data were extensively filtered by joint mapping to the transcriptome and ribosomal RNA using QuickMIRSeq. Sequences were aligned to the reference genome GRCh38.p13. The count data were transformed to log2-counts per million (logCPM) using the voom function from the limma package [16 (link)] in R. Differential expression analysis was performed utilizing the limma package. A false positive rate of α = 0.05 with a false discovery rate (FDR) correction was considered as the level of significance. Volcano plots and heatmaps were created using ggplot2 package (version 2.2.1) and the iDEP [17 (link)](version 0.91).

Total RNA was extracted from approximately 1 × 10⁶ cells using a Qiagen AllPrep RNA isolation kit (Qiagen, Limburg, Netherlands) according to the manufacturer’s instructions. The concentration and purity of the extracted RNA were determined using Nanodrop (Agilent Technologies, Santa Clara, CA, USA). The RNA samples were stored at −80°C for later use. Complementary DNA (cDNA) was synthesized from 3 µg of total RNA using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche Applied Science, Mannheim, Germany) using a random hexamer primer and an anchored-oligo (dT) primer. Expression of selected genes was analyzed using an ABI StepOnePlus™ Real-Time PCR machine (Applied Biosystems, Foster City, CA, USA). The qPCR reaction was prepared using a Taqman^® gene expression assay (Applied Biosystems) and the primers listed in Table 2.