Characterizing the salivary RNA landscape to identify potential diagnostic, prognostic and follow-up biomarkers for breast cancer
Characterizing the salivary RNA landscape to identify potential diagnostic, prognostic and follow-up biomarkers for breast cancer
Unstimulated saliva samples were collected from breast cancer (BC) patients and healthy individuals using DNA Genotek collection tubes (Ref CP-190). Participants were instructed to avoid eating, drinking, smoking, or oral hygiene activities for at least 30 minutes before collection to reduce variability in saliva composition. Each saliva-filled tube was shaken vigorously for at least 8 seconds to ensure sample homogeneity.  In their original collection vials, samples were incubated at 50°C for 1 hour in a water bath. After incubation, samples were aliquoted into 1 ml portions in separate microcentrifuge tubes for storage at –80°C. Saliva samples were thawed on ice, and a 0.5 mL aliquot was incubated at 90°C for 15 minutes to inactivate RNases, then cooled to room temperature. A neutralizer solution (20 μL, 1:25) was added to the saliva aliquot, followed by a 10-minute incubation on ice. Samples were then centrifuged at maximum speed for 3 minutes. The supernatant was carefully transferred to a fresh 5 mL tube without disturbing the pellet. The samples were then stored at -80 awaiting further RNA extraction. RNA was isolated from cell-free saliva samples using the miRNeasy Serum/Plasma Advanced Kit (Qiagen Cat no: 217184), following the manufacturer's protocol with slight modifications. To each sample, 2.5 mL of QIAzol lysis reagent was added, and the mixture was vortexed to ensure thorough lysis, followed by a 5-minute incubation at room temperature. Tubes were capped, vortexed for 15 seconds, incubated at room temperature for 2–3 minutes, and centrifuged at 12,000 x g at 4°C for 15 minutes. The upper aqueous phase was carefully transferred to a new 5 mL tube, avoiding contamination from the interphase, and mixed with 1.5 volumes of 100% (final volume ~5 mL). For RNA purification, the sample was loaded in 700 μL aliquots, onto an RNeasy MinElute spin column connected to a Vac-Man® Laboratory Vacuum Manifold. The vacuum was opened carefully. The process was repeated until the entire sample was loaded. The column was then removed from the vacuum manifold, placed in a 2 mL collection tube, and washed with 700 μL Buffer RWT, followed by centrifugation at ≥8000 x g for 15 seconds. The column was subsequently washed with 500 μL Buffer RPE and centrifuged at ≥8000 x g for 15 seconds, followed by a final wash with 500 μL of 80% ethanol and centrifugation at ≥8000 x g for 2 minutes at room temperature. The column was transferred to a fresh 2 mL collection tube with the lid open and centrifuged at full speed for 5 minutes to dry the membrane. Finally, the column was placed into a new 1.5 mL collection tube, and RNA was eluted by applying 30 μL of RNase-free water directly to the center of the membrane, followed by centrifugation at full speed for 1 minute.  RNA sequencing was performed at Novogene (Cambridge, United Kingdom). First-strand cDNA synthesis was carried out using random hexamer primers, followed by second-strand cDNA synthesis using either dUTP for directional libraries or dTTP for non-directional libraries.