Biotek synergy neo multi

Ambion RNaseAlert Lab Test kit (Thermo Fisher Scientific, #AM1964) was used to detect RNase enzymatic activity according to the manufacturer’s instruction. Briefly, 5 μl of 10-fold RNaseAlert buffer was added to a tube containing the fluorescent substrate, and then mixed with a total of 45 μl of the tested CM with 1/1000 dilution by RNase-free water to reduce background RNase activity. The mixture was sequentially placed on a well of a 96-well plate. The real-time fluorescence data were collected at 1 min intervals for 21 min using a BioTek Synergy™ Neo multi-mode reader (BioTek Instruments).

Serum collection from breast cancer patients and healthy individuals was approved by Institutional Review Board of MD Anderson Cancer Center and informed consent was obtained from all subjects. The concentration of hRNase 1 in serum or CM was determined by ELISA Kit for hRNase 1 (Cloud-Clone Corp., #SEA297Hu) according to the manufacturer’s instructions. Briefly, standards or samples were added to the appropriate wells of a pre-coated and ready-to-use 96-well plate for 1 h at 37 °C, followed by incubating with a biotin-conjugated antibody against hRNase 1 (Detection Reagent A) for 1 h at 37 °C. After washing, horseradish peroxidase (HRP)-conjugated avidin (Detection Reagent B) was added to each well, and the mixture was incubated for 1 h at 37 °C. After washing, 90 µl of TMB substrate solution was added to each well for 10–20 min at 37 °C. Finally, the enzyme–substrate reaction was stopped by 50 µl of sulfuric acid solution. Only the well added with hRNase 1, biotin-conjugated antibody, and HRP-conjugated avidin displayed a change of color, which was subsequently measured at a wavelength of 450 nm by a BioTek Synergy™ Neo multi-mode reader (BioTek Instruments). The concentration of hRNase 1 in the samples was calculated by comparing the optical density value of the samples to the standard curve.