disease state: normal control sample type: saliva supernatant gender: male age (year): 48
Treatment protocol
Subjects were asked to refrain from eating, drinking, smoking, and oral hygiene procedures for at least 2 hours before the collection. To stimulate glandular salivary flow, subjects received a citric acid solution for application to the surface of the tongue with a cotton swab for 5 s every 30 s. The citric acid stimulation continued at 30-second intervals during the entire collection procedure. Up to 3 mL of saliva from each subject was collected in a 50-mL centrifuge tube. Saliva samples was centrifuged at 3,000 × g for 15 min at 4°C to spin down exfoliated cells, and the supernatant was transferred into microcentrifuge tubes followed by a second centrifugation at 12,000 × g for 10 min at 4°C to completely remove cellular components. Samples were stored at -80°C until use.
Extracted molecule
total RNA
Extraction protocol
1)Using the mirVana PARIS kit (Ambion, USA),1.2ml of saliva was distributed equally to 2 microcentrifuge tubes.That is to say, each microcentrifuge tube contains 600μL of saliva. 2)200μL of 2x denaturing solution was added to each tube. 3) Add 0.8ml of Acid-Phenol:Chloroform to each tube, and vortex for 30–60 sec to mix. 4) Centrifuge for 5 min at maximum speed (≥10,000×g) at room temp to separate the mixture into aqueous and organic phases. After centrifugation, the interphase should be compact; if it is not, repeat the centrifugation. 5) Carefully remove the aqueous (upper) phase without disturbing the lower phase or the interphase, and transfer it to a fresh tube. 6) Add 1.25 volumes 100% ethanol to the aqueous phase, and mix thoroughly. 7) For each sample, place a Filter Cartridge into one of the Collection Tubes. Pipet the lysate/ethanol mixture onto the Filter Cartridge. And apply the mixture in successive applications to the same filter. Centrifuge for ~30 sec or until the mixture has passed through the filter. Discard the flow-through, and repeat until all of the lysate/ethanol mixture is through the filter. Save the Collection Tube for the washing steps. 8) Wash the filter with 700 μLWash Solution 1. 9) Wash the filter twice with 500 μL Wash Solution 2/3. 10)RNA in the filter was eluted in 30μL of preheated nuclease-free water(95℃)
Label
Cy3
Label protocol
miRNA molecular in total RNA was labeled by miRNA Complete Labeling and Hyb Kit(Cat#5190-0456, Agilent technologies, Santa Clara, CA, US)followed the manufacturer’s instructions, labeling section.
Hybridization protocol
Each slide was hybridized with 100ng Cy3-labeled RNA using miRNA Complete Labeling and Hyb Kit(Cat#5190-0456, Agilent technologies, Santa Clara, CA, US)in hybridization Oven(Cat#G2545A, Agilent technologies, Santa Clara, CA, US)at 55℃，20rpm for 20 hours according to the manufacturer’s instructions, hybridization section. After hybridization, slides were washed in staining dishes (Cat#121, Thermo Shandon, Waltham, MA, US) with Gene Expression Wash Buffer Kit(Cat#5188-5327, Agilent technologies, Santa Clara, CA, US).
Scan protocol
Slides were scanned by Agilent Microarray Scanner(Cat#G2565BA，Agilent technologies, Santa Clara, CA, US)and Feature Extraction software 10.7 (Agilent technologies, Santa Clara, CA, US)with default settings.
Description
microRNA expression profile of saliva from healthy control No.1
Data processing
Raw data were normalized by Quantile algorithm, Gene Spring Software 11.0(Agilent technologies, Santa Clara, CA, US).
