Before DNA isolation, pelleted A. marginale omp10:: himutantstants were treated with RNaseA (QIAGEN) and DNase I (Sigma Aldrich) to remove ISE6 host cell contaminant nucleic acids.
For microRNA isolation, pelleted cells were resuspended in TRIzol reagent (Invitrogen, Carlsbad, CA, USA) (which retains small RNA molecules), subjected to polyethylene glycol precipitation, RNA ligase-mediated labeling, and hybridized to a custom array, according to previously published method [ 56].
For mRNA isolation pellets were transferred into 500 μl of RA1 lysis buffer (NucleoSpin RNA; Macherey & Nagel, Düren, Germany) containing 5 μl β-mercaptoethanol and homogenized by pipetting.
For DNA isolation, the pellet was resuspended in 400 μl TE buffer pH 7.0.
After a brief centrifugation at 600 g in the cold isolation buffer, pellet and supernatant were combined, rehomogenised, and centrifuged at 800 g for 10 minutes at 4°C.
The epidemiologic investigation also concluded that virus isolation from pellets would be critical evidence that the pellets caused the outbreak.
For RNA isolation, rickettsial pellets were solubilized using 1 ml of Tri-Reagent (Applied Biosystems, Austin, TX) per-flask equivalent for L929-grown rickettsiae or per 0.5 ml of hen egg yolk sac rickettsial preparations.
Before RNA isolation, the pellets were collected in a 1.5 ml micro-tube (Sahrstedt, Nümbrecht, Germany) containing RLT buffer (Qiagen) and disrupted by sonication.
Briefly, 5 × 10 cells were used for mitochondrial isolation and pelleted mitochondria were boiled in lysis buffer for 3 min to yield mitochondrial lysate.
For quantification of mitochondrial protein, an aliquot was removed, centrifuged at 8,000 g for 15 min, washed in BSA-free isolation buffer, pelleted, and used for determination of protein concentration.
