Ribopure blood rna purification kit

Blood from 10 healthy donors (five men and five women; age range: 35–60 years) was collected and exposed to a range of X-ray doses (0.25, 0.5, 1 and 2 Gy at a dose rate of 0.5 Gy min⁻¹) and after 24 h, RNA was extracted using a RiboPure™-Blood RNA Purification Kit (Thermo Fisher Scientific, Loughborough, UK) and the cDNA synthesized using High Capacity cDNA reverse transcription kit (Applied Biosystems, FosterCity, CA, USA). The cDNA from the 10 donors was combined and used as a calibration curve in each multiplexed QRT-PCR run to estimate the dose of the blood samples stimulated with LPS and curcumin as previously described (Manning et al. 2013 (link)). A linear fit was used to construct the dose estimation curve and the increase in expression following irradiation for each sample was entered into the linear equation to give a dose estimate.

Total RNA from blood samples exposed ex vivo to X-rays was extracted using a RiboPure™-Blood RNA Purification Kit (Thermo Fisher Scientific, Loughborough, UK). Total RNA from samples collected in PAXgene tubes from radiotherapy patients was extracted with the PAXgene Blood miRNA kit (Qiagen, PreAnalytiX GmbH, Hilden, Germany) using a robotic workstation Qiacube (Qiagen, Manchester, UK). The quantity of isolated RNA was determined by spectrophotometry with a ND-1000 NanoDrop and quality was assessed using a Tapestation 220 (Agilent Technologies, CA, USA). cDNA was prepared from 350 ng of the total RNA using High Capacity cDNA reverse transcription kit (Applied Biosystems, FosterCity, CA, USA) according to the manufacturer’s protocol.
For samples exposed to gamma-radiation, total RNA was extracted from 400 μL of whole blood with the Nucleospin RNA Blood kit (Macherey and Nagel, Hoerdt, France) according to the manufacturer’s instructions. RNA was converted into cDNA using the Enhanced Avian HS RT-PCR Kit (Sigma-Aldrich, Lyon, France) with oligo-dT priming according to the manufacturer’s protocol.