The quality (defined as salivary protein content, viscosity, pH and buffer capacity) and the quantity of saliva (mostly related to flow rate) play a crucial role in the equilibrium between demineralization and remineralization of enamel in a cariogenic environment [1].
Total RNA was eluted in 30 μl ice-cold TE buffer and the RNA quantity and quality were assessed using a NanoDrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, USA) and Experion RNA Stdsens analysis kit (Biorad Laboratories Inc., USA), respectively.
The suspensions of fungal spores were prepared after 7 10 days of fungi growth, until full sporulation, after which the spores were collected by plastic microbiological loop in 100 mM phosphate buffer (pH 6.5), and the quantity of spores was determined in counting chamber "Malassez"(Germany).
The total RNA was resuspended in TE buffer (pH 7.4) and the quantity and quality of the RNA aliquots were checked on a bioanalyzer (Agilent Technologies).
The RNA was eluted in Elution Buffer, and RNA quantity and quality were checked using the RNA Pico-Chip on the Bioanalyzer 2100 (Agilent Bioscience, Mt View, CA).
Finally, binding buffer (300 μL) was added to each tube, and the quantity of apoptotic cells in samples was analyzed by flow cytometry (FC500, Beckman Coulter, USA).
DRG were lysed in RLT® buffer using a tissue lyser (Qiagen Doncaster, Australia), RNA was extracted (RNeasy Mini-kit, Qiagen) and the quantity and quality of the RNA determined (Nanodrop, Thermo Scientific, USA, Bioanalyzer, Agilent, USA).
The integrity of the total RNA was checked by electrophoresis in 1% agarose gel in Tris-acetate-EDTA (TAE) buffer and quantity was determined with a NanoVue™ Spectrophotometer (GE healthcare).
The sudden news -- and the quantity!
Briefly, pooled serum from a large quantity of mice was purified using saturated ammonium sulfate buffer and the concentration of total serum proteins in stock for all of the studies was measured and adjusted to 20 mg/ml in saline.
