le> Materials and methods Sample collection and preparation
ssDNA from herring sperm (Sigma, St Louis, MO) was used as the ssDNA standard. Calf liver 18S + 28S ribosomal RNA (rRNA; Sigma) was used as control RNA. Spiked-in samples were prepared as follows: 8µg of herring sperm DNA or calf liver 18S + 28S rRNA was added to 50µL of BSA 1mg/ml in PBS; 5µg of herring sperm DNA was added to 50µL of HeLa (BD, Franklin Lakes, NJ, USA) and Jurkat + CalyculinA (Cell Signaling Technology, Danvers, MA, USA) commercial cell lysates (0.5mg/ml protein concentration); 2µg of herring sperm DNA was added to 50µL of lymph node (lymph node metastasis from primary breast cancer) sample lysate and 970073 and 040102 (bone metastasis from primary lung and renal carcinomas, respectively) sample lysates (unknown protein concentration). An equal amount of each sample was used to prepare the RPMAs.
RPMI 8226 and U266 cell lines (ATCC, Manassas, VA, USA) were used to create cell lysates from a specific number of cells. RPMI 8226 and U266 cells were maintained as suspension cultures in RPMI 1640 medium (ATCC) at 37°C, 5% CO2, and 70% humidity. Cells were counted in a hemacytometer, then a known number of cells were removed from culture and lysed with protein extraction buffer: 45% T-PER (Pierce, Rockford, IL), 45% Novex Tris-Glycine SDS Sample Buffer (2X) (Invitrogen, Carlsbad, CA), 10% TCEP Bond Breaker (Pierce), and heated at 100°C for 5 min.
Peripheral blood for preparing enriched RBC samples was obtained by venipuncture from a healthy volunteer with informed consent. EDTA anti-coagulated peripheral blood was spun twice at 200× g for 10 min. The buffy coat and plasma were discarded after each centrifugation step to enrich the RBC fraction. RBCs were counted in a hemacytometer and a known number of cells were incubated in protein extraction buffer for 10 min then heated at 100°C for 5 min (RBC lysate). Mixtures of RPMI 8226 cells and RBCs were prepared by mixing a known number of cells of each type in the ratio of 10:1 and 1:10. Cells were lysed in protein extraction buffer for 10 min then heated at 100°C for 5 min.
Bone metastasis and normal muscle tissue samples were collected at the Istituto Ortopedico Rizzoli (IOR), IRCCS, Bologna, Italy, under an IRB-approved protocol with informed consent. Specimens were snap-frozen and maintained at -80°C. Samples were placed in protein extraction buffer and lysis was performed using Adaptive Focus Acoustic (AFA) technology (Covaris) at 20% duty factor, 275 peak incident power, and 200 cycles per burst for 90 s.RPMA construction
RPMAs were printed with whole cell lysates, ssDNA, and RNA controls, in duplicate or triplicate. Lysates were printed on glass-backed nitrocellulose array slides (SCHOTT Nexterion, Germany) in a 2-fold dilution series using an Aushon 2470 arrayer equipped with 350µm pins (Aushon Biosystems, Billerica, MA, USA). After printing, the slides were either baked for 2 h at 80°C and then stored, or stored without baking, with desiccant (Drierite, W. A. Hammond, Xenia, OH, USA) at -20°C prior to use.
Slides were treated with ReBlot mild solution (Millipore, Billerica, MA, USA) for 15 min and washed twice in PBS. The slides were blocked (I-Block, Applied Biosystems) for 2 h before immunostaining. Immunostaining was performed on an automated slide stainer according to the manufacturer's instructions (Autostainer CSA kit, Dako, Carpinteria, CA, USA). Each array was probed with a single polyclonal or monoclonal primary antibody (Table 1) for 30 min. Negative control slides were incubated with antibody diluent (Dako). Secondary antibody was goat anti-rabbit IgG H + L (1:7500) (Vector Laboratories, Burlingame, CA, USA) or rabbit anti-mouse IgG (1:10) (Dako) (5, 12, 13). Subsequent protein detection was performed with diaminobenzidine according to the manufacturer's instructions (Dako).Total protein microarray staining
Total protein staining was performed using Sypro Ruby Protein blot stain (Invitrogen) according to the manufacturer's instructions and scanned with a NovaRay CCD imager (Alpha Innotech, San Leonardo, CA, USA) equipped with a Cy3 filter.Imaging and data analysis
The immunostained slides were scanned on a UMAX 2100XL flatbed scanner using the following settings: white balance 255, black 0, middle tone 1.37, 600dpi, 14 bit. Spot intensity was analyzed by Image Quant v5.2 software (Molecular Dynamics). Data reduction was performed with a VBA Excel macro, RPMA Analysis Suite (RAS) (http://capmm.gmu.edu/rpma-analysis-suite). To normalize data, the relative intensity value for each endpoint for each spot was divided by the relative intensity value for the selected normalization molecule (or the geometric mean of a group of analytes) for the corresponding spot.