Qiaamp 96 qiacube ht kit

Genomic DNA or total RNA were extracted from flash frozen tissues with a QIAcube HT (Qiagen, Germany) using the QIAamp 96 QIAcube HT kit or RNeasy 96 QIAcube HT kit. In brief, two small samples from each frozen tissue (approx. 30 mg) were cut and placed in separate, 2 mL Eppendorf tubes. Each tube held a 5 mm stainless-steel bead and either 700uL of QIAzol lysis buffer or 450uL of ATL buffer for RNA and DNA extraction, respectively. The tubes for total RNA extraction were then shaken twice at 25 Hz for 5 minutes using the Qiagen Tissue Lyser II (Qiagen, Germany). After lysis, 200uL of chloroform was added and manually shaken. Samples were incubated for 3 minutes at RT and then centrifuged for 15 minutes at 4 °C. After centrifugation, the aqueous phase was isolated and transferred to a deep-well, 96-well plate and total RNA was isolated using the QIAcube HT system. For DNA extraction, the tubes with tissue and ATL buffer were shaken twice for 1 minute at 25 Hz in the Qiagen Tissue Lyser II. 50uL of proteinase K was added to each sample and then samples were incubated overnight at 56 °C with shaking. Genomic DNA was extracted using the QIAcube HT system.

The 10-g cheese slices were treated as described by Zago et al. [18 (link)]. Briefly, the samples were homogenized twice in a Stomacher 400 Circulator (Seward Laboratory, London, UK) with sterile sodium citrate (NaCt 2% w/v, pH 7.5). The cheese homogenate was centrifuged (14,400× g for 7 min at 4 °C) for fat removing. The pellet was then resuspended in Triton X-100 (2.5% v/v), washed twice with phosphate-buffered saline (pH 7.5), and centrifuged (8700× g, 7 min, 4 °C). Finally, after the pellet was resuspended in Tris-EDTA (0.1 M, pH 8), total dsDNA was extracted using a QIAcube HT automated station (Qiagen, Milan, Italy) using QIAamp 96 QIAcube HT kit (Qiagen, Milano, Italy). Total dsDNA was quantified fluorometrically (Qubit^TM, Life Technologies, Monza, Italy).