Dnase type 1 ambion turbo dna free kit

Total RNA was isolated from 100 mg of lung, tracheobronchial lymph node, and thymus homogenized with 2 ml of TRIzol™ LS Reagent using homogenizer 150 (FisherBrand™, Thermo Fisher Scientific) and the NucleoSpin^® RNA Virus Column kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s protocols. In order to remove genomic DNA, a DNase type I Ambion^® TURBO-DNA-free™ kit (Life Technologies, Carlsbad, CA, USA) was applied following the manufacturer’s instructions. The concentration and purity of the extracted RNA were determined by spectrophotometry using the Nanodrop 2000 (Thermo Fisher Scientific). One microliter of total RNA was used to generate cDNA using the Script™ cDNA Synthesis Kit (BioRad, Hercules, CA, USA) following the manufacturer’s indications.

RNA was isolated from a total of 100 mg of lung (around 33 mg per lobule, cranial, middle and caudal) and tracheobronchial lymph node tissue samples separately homogenized with 2 ml of TRIzol™ LS Reagent using a homogenizer 150 (FisherBrand™Thermo Fisher Scientific, Barcelona, Spain), and following the manufacturer’s guidelines. Then, RNA was extracted by using the NucleoSpin® RNA virus columns kit (Macherey-Nagel, Düren, Germany) following manufacturer’s instructions. In order to remove genomic DNA, the DNase type I Ambion® TURBO-DNA-free™ kit (Life Technologies, Carlsbad, CA, USA) was used. A determination of concentration and purity of isolated RNA was conducted by Nanodrop 2000 (Thermo Fisher Scientific, Barcelona, Spain) obtaining samples with a ratio 260/280 around 2. Finally, we used 1 μl of high-quality purified RNA to generate cDNAs by Script™cDNA Synthesis Kit (BioRad, Hércules, CA, USA) following manufacturer’s specifications.