Nano drop nd 100 spectrophotometer

Only male H. anatolicum were selected for DNA extraction due to an insufficient number of female ticks. Before DNA extraction, ticks were washed in 70% ethanol and deionized water for 5 min to remove environmental contaminants in accordance with a published protocol [23 (link)]. Due to the small size of each tick, a pool of five male H. anatolicum ticks was homogenized in liquid nitrogen inside a sterile 1.5-ml microcentrifuge tube using a sterile Kimble Kontes pellet pestle (Thermo Fisher Scientific, Waltham, MA, USA). Genomic DNA was extracted from each pool using the QIAamp Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. The concentration and quality of DNA were assessed using a Nano Drop ND-100 spectrophotometer (Peqlab Biotechnologie GmbH, Erlangen, Germany). DNA quality was also determined by electrophoresis in a 1% agarose gel; the bands were stained with ethidium bromide and visualized under UV light. DNA was stored at − 20 °C in the freezer until further use. Prior to sequencing, extracted DNA samples (5 DNA samples of ticks from each host) were pooled again to make one pool for each host from each location, resulting in seven DNA pools.

TNAs were extracted from immature leaves (10–20 mm in length) using a phenol-chloroform-based method [49 (link)] and quantified using a NanoDrop ND-100 spectrophotometer (Peqlab GmbH, Erlangen, Germany). Virus accumulation was evaluated through Southern blot analyses after separating 500 ng of TNAs per lane in 1% agarose gels in TBE, either in the presence (Figure 1) or absence (Figure 2) of 0.5 μg/mL ethidium bromide. TNAs transferred onto nylon membranes were hybridized with DNA probes specific for either the full-length AbMV DNA-A or the TYLCSV CP gene sequence, respectively, as described by [49 (link),50 (link)]. Probes were labelled with digoxigenin via a DIG-High Prime kit and detected through CSPD or CDP Star chemiluminescence, following the manufacturer’s instructions (Roche Deutschland Holding GmbH, Penzberg, Germany). Signals were visualized on X-ray films (Fujifilm, Reutlingen, Germany) and documented using a transmitted light scanner.

Total RNA was isolated from snap-frozen, murine lungs using the Nucleo Spin RNA/Protein isolation Kit (Macherey–Nagel, Düren, Germany), quality checked and quantified using the NanoDrop ND-100 Spectrophotometer (Peqlab, Wilmington, USA). Transcript expression levels of murine Clca3, Clca5, Muc5ac and Muc5b, normalized to the reference genes elongation factor 1α (Ef-1α), β-2 microglobulin (B2m) and glyceraldehyde-3-phosphate dehydrogenase (Gapdh), were determined as described (Dietert et al. 2014 (link)). RT-qPCR and data analyses were conducted using the CFX96 Touch Real-Time PCR Detection System and CFX Manager software 1.6 (BioRad). Relative quantification and comparison of groups were performed by the ΔΔCt method using naive animals as controls.