Nanodrop nd 1000 uv vis spectrophotometer

Brains of wild-type C57BL/6N mice were dissected within 24 h after birth (postnatal days 0–1), and the cortices were isolated, transferred into HBSS-containing tubes, and treated with PDD solution (HBSS -/-, 0.01% papain (w/v) (Worthington Biochemical Corporation, Lakewood, NJ, USA), 0.1% (w/v) dispase II (Hoffmann-La Roche AG, Basel, Switzerland), 0.01% (w/v) DNase I (Worthington Biochemical Corporation), 12.4 mM MgSO₄) [54 (link)], followed by mechanical trituration. A total of 2 × 10⁵ cells per well were seeded in an NBA-media mix (Neurobasal A, 1% (v/v) GlutaMax, 2% (v/v) B27 50x, 1% (v/v) sodium pyruvate, 1% (v/v) antibiotic/antimycotic (all from Gibco/Thermo Scientific, Schwerte, Germany)) in 12-well plates coated with poly-L-lysine (2.5 mg/mL poly-L-lysine (Sigma-Aldrich, Munich, Germany) in 150 mM borate buffer pH 8.4). Cells were cultured for one week at 37 °C in a 5% CO₂ humidified atmosphere and treated as indicated with 10 µM fluoxetine [15 (link)].
RNA isolation was performed with the Purelink RNA Kit from Thermo Scientific (Schwerte, Germany), according to the manufacturer’s protocol. RNA qualities and concentrations were assessed using a NanoDrop ND-1000 UV-Vis spectrophotometer (Peqlab, Erlangen, Germany). Five hundred nanograms of RNA were reverse transcribed into cDNA using the Quanta cDNA Kit (Gaithersburg, MD, USA), according to the manufacturer’s protocol.

Samples of embryos, pre-larvae and larvae were let to thaw on ice, disrupted and homogenized using the TissueRuptor (Qiagen, Hilden, Germany) for 20 s in 600 μl RLT plus buffer (RNeasy Plus Mini Kit Qiagen, Valencia, USA). Total RNA was isolated with the RNeasy Plus Mini Kit (Qiagen, Valencia, USA). RNA yield and purity was determined by measuring the absorbance at 260 and 280 nm using a Nanodrop^® ND-1000 UV–Vis spectrophotometer (Peqlab, Erlangen, Germany), and its integrity was tested by electrophoresis in 1% agarose gels. Reverse transcription (RT) was carried out using QuantiTect Reverse transcription kit (Qiagen, Valencia, USA) using 1 μg of total RNA, according to the manufacturer’s instructions.

For the Hannover cohort, macrodissected FFPE GBM tissues were deparaffinized with xylene and genomic DNA (gDNA) was isolated using the QIAamp DNA FFPE Tissue kit (Qiagen, Hilden, Germany). The gDNA concentration and purity was assessed using a NanoDrop ND-1000 UV-VIS spectrophotometer (PeqLab, Erlangen, Germany). The isolated gDNA was subjected to bisulfite conversion using the EpiTect Bisulfite Kit (Qiagen) followed by methylation-specific polymerase chain reaction (MSP) using the primer sets established by Esteller et al. [25 (link)]. EpiTect bisulfide converted unmethylated and methylated human DNAs (Qiagen) as well as samples without any DNA were included as controls. The PCR products were separated by electrophoresis on 3% agarose gels. Gel imaging and analysis was performed with BioDocAnalyse Sytems (Biometra, Göttingen, Germany). The tumor specimens which showed a visible band of methylated PCR product were considered as methylated, whereas the absence of this band indicated unmethylated tumor samples.
For the Bologna cohort, the MGMT promoter methylation analysis was performed exactly as described previously [26 (link)].

Head kidney samples (20–30 mg) were disrupted and homogenized using the TissueRuptor (Qiagen, Hilden, Germany) for 20 s in 350 μl LBP lysis buffer (NucleoSpin^® RNA Plus, MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany). Total RNA was isolated using the NucleoSpin^® RNA Plus kit (MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany), according to manufacturers’ instructions. RNA yield and purity were determined by measuring the absorbance at 260 and 280 nm, using Nanodrop^® ND-1000 UV-Vis spectrophotometer (Peqlab, Erlangen, Germany), while its integrity was tested by 1% agarose gel electrophoresis. Reverse transcription (RT) was performed using 1 μg RNA with the QuantiTect Reverse transcription kit (Qiagen, Valencia, USA), following manufacturer’s instructions. One LR and one HR samples were excluded from the analysis due to insufficient RNA yield and purity.

Fasting blood samples for RNA extraction were drawn in PAXgene TM Blood RNA tubes (Qiagen, Hilden, Germany) and stored at −80 °C; RNA was isolated according to the manufacturer’s instructions. The concentration of RNA was determined photometrically using a NanoDrop ND-1000 UV-Vis spectrophotometer (Peqlab, Erlangen, Germany). Five hundred nanograms of RNA were used in a 20 μL reverse transcription reaction using the Quanta cDNA Kit (Gaithersburg, MD, USA) to synthesize cDNA.

RNA and protein were isolated in parallel from the same sample using NucleoSpin RNA/Protein kit (Macherey–Nagel, Düren, Germany) according to the instruction of the manufacturer. The isolated RNA was quantified by NanoDrop ND-1000 UV–Vis Spectrophotometer (peqLab Biotechnologie GmbH, Erlangen, Germany). The concentration of protein was determined using bicinchoninic acid (BCA) protein assay.