Home » Protocols » Processing of u…
Protocol
Processing of urine and buccal cells
Keywords: urine, buccal, cell sample
The protocol is derived from the WHO/IARC guideline “Common Minimal Technical Standards and Protocols”. The purpose of the document is to provide a framework for the development and coordination of Biological Resource Centers.
Required Materials
- Cryovial
- Vacutainer , or similar container with a 50-3000 ml capacity
- Liquid Nitrogen , for storage
- Tris-EDTA buffer
Instructions
-
Urine
- Plastic or glass containers for collection of urine should be clean and dry, have a 50-3000ml capacity, wide mouth and leak-proof cap, and should be clearly labelled.
- When in transit, urine collections should be maintained on ice or refrigerated.
- Urine should be aliquoted according to the volume necessary for analysis or storage.
- Depending on the analyte to be measured, already during collection or before aliquoting a preservative may be added.
- Store urine at -80°C or lower in liquid nitrogen.
-
Buccal Cell
- A collection kit (containing mouthwash, 50ml plastic tube, plastic biohazard bottle, and courier packaging) is mailed or given to the participant, along with an instruction sheet. The participant is to brush the teeth as usual, rinse the mouth well twice with water, and then wait 2 hours. They should not eat or drink anything other than water during this time.
- After 2 hours, 10ml of commercial mouthwash should be poured into the tube, and then 10ml tap water added. This diluted mouthwash should be placed into the mouth (without swallowing) and swished around vigorously for 30 seconds.
- The mouthwash should then be spat back into the plastic tube and the tube should be sealed tightly.
- The sample should be sent back to BRC immediately for processing, or stored at 4°C until sent but should be sent within 24 hours.
- On arrival at the laboratory, transfer mouthwash to 15ml conical test tubes.
- Add 35ml Tris-EDTA to the mouthwash sample and spin at 450g for 5 minutes.
- Decant supernatant and discard.
- Wash cells twice, each time with 45ml Tris-EDTA.
- Resuspend cell pellet in 50μl Tris-EDTA and transfer to 2ml labeled cryovials.
- Store sample at -80°C or in liquid nitrogen. Note: the collection of buccal cells can also be conducted with other means such as brushes for example.
-
Saliva (1BXQD00)
A UCLA research consortium was funded by the National Institute of Dental and Craniofacial Research (NIH grant UO1 DE 16275; PI: David T. Wong DMD, DMSc) to investigate the human saliva proteome. The protocol for saliva collection and processing is derived from their “Salivary Proteome Handbook Procedures and Protocols”.
-
Collecting salivary samples
Saliva collections is recommended to be done in the morning (please aim for 10-11am if possible), and ask the subject to refrain from eating, drinking, or oral hygiene procedures for at least 1 hour prior to the collection.
The subject will be given drinking water (bottled) and asked to rinse their mouth out well (without drinking the water).
5 minutes after this oral rinse, the subject will be asked to spit whole saliva (WS) into a 50ml sterile Falcon® tube. The subjects need to refrain talking. It is better for subject to drop down the head and let the saliva run naturally to the front of the mouth, hold for a while and spit into the tube provided. Subjects will spit into the collection tube about once a minute for up to 10 minutes. The goal for each whole saliva donation should be about 5ml. Require that the tube is placed on ice while collecting whole saliva. CROTICAL: Remind the subjects not to cough up mucus as saliva is collected, not phlegm.
COLECTED SAMPLES ARE TO BE KEPT ON ICE AT ALL TIMES PRIOR TO PROCESSING.
-
Submandibular saliva (SM) collection
Use 2 x 2 cotton gauze to block the opening of each parotid duct; dry up the floor of the mouth and block the openings of the sublingual gland (both sides) and have the subject raise their tongue slightly to elevate the opening to the SM gland. Begin to collect SM saliva by using sterilized Wolf device. A sterilized and disposable yellow tip (for pipette P200) is connected into the device and changed between every collection. During the collection at 2 minute intervals, a few grains of citric acid powder are swabbed with a moistened cotton applicator onto the lateral dorsum of the tongue to stimulate the secretion. Aim to collect at least 200μL SM.
-
Sublingual saliva (SL) collection
Similar to the protocol described above for SM collection. The only difference is that the ductal orifices of the submandibular gland will be blocked off. Aim to collect > 100μL SL every time.
-
Parotid saliva (PR) collection
Use Parotid cup to collect PR. Parotid cups may be placed bilaterally if the clinical investigator so chooses. This will allow for the simultaneous collection from each parotid gland. The citric acid stimulation will be performed as described above. Aim to collect > 1mL PR.
The first 0.1ml of parotid saliva collected will be discarded to insure that fresh parotid saliva is obtained.
-
Sample processing using proteinase inhibitors
To each 100μl saliva, add:
- 0.2μL proteinase inhibitor cocktail from standard stock solution (Sigma, cat# P8340), invert gently.
- 0.3μL Na3OV4 (Sigma, cat# S6508) from standard stock of 400mM, invert gently.
Centrifuge specimens at 2600xg for 15 minutes at 4°C; (if you note that incomplete separation has occurred, you may increase the spin time to 20 minutes). Remove the supernatants from the samples and mark them as plus “super” which stands for the supernatant phase of the saliva. Taking care not to disturb the pellet and please keep the pellet as is in the original tubes, you may like to add marks to the original tubes with “pellet”.
Freeze samples at -80°C.
-
Collecting salivary samples
Saliva collections is recommended to be done in the morning (please aim for 10-11am if possible), and ask the subject to refrain from eating, drinking, or oral hygiene procedures for at least 1 hour prior to the collection.
The subject will be given drinking water (bottled) and asked to rinse their mouth out well (without drinking the water). 5 minutes after this oral rinse, the subject will be asked to spit whole saliva (WS) into a 50ml sterile Falcon® tube. The subjects need to refrain talking. It is better for subject to drop down the head and let the saliva run naturally to the front of the mouth, hold for a while and spit into the tube provided. Subjects will spit into the collection tube about once a minute for up to 10 minutes. The goal for each whole saliva donation should be about 5ml. Require that the tube is placed on ice while collecting whole saliva. CROTICAL: Remind the subjects not to cough up mucus as saliva is collected, not phlegm.COLECTED SAMPLES ARE TO BE KEPT ON ICE AT ALL TIMES PRIOR TO PROCESSING.
-
