Saliva collection, processing, stabilization, and storage method
View Patent ↗Provided herein is an all-in-one saliva collection apparatus that collects saliva to allow for the filtration of saliva in order to separate saliva components, such as extracellular proteins and nucleic acids that are not present in intact cells, from the intact cells and debris remaining in the extracted sample. The filtered saliva samples can be aliquoted into two fractions for protein and/or nucleic acid analysis. The present invention further describes long term storage at ambient temperatures of filtered salivary nucleic acids, and long term storage at ambient temperatures of filtered salivary proteins added to an ethanol solution. The filtered cell-free saliva samples have diagnostic usefulness.
1. A method for stabilizing nucleic acid and protein samples isolated from a saliva sample, the method comprising:
a) collecting a saliva sample from a subject;
b) filtering the saliva sample at ambient temperature to produce a filtered sample that is free of cells;
c) collecting the filtered sample at ambient temperature into at least a first and a second receiving device;
d) adding an alcohol solution to the first receiving device at ambient temperature to produce an alcohol-containing filtered sample to form a protein sample, with the proviso that alcohol is not added to the second receiving device to produce an alcohol-free filtered sample to form a nucleic acid sample; and
e) storing the protein sample and the nucleic acid sample for at least 3 days at 25 degrees Celsius.
2. The method of claim 1 , wherein the nucleic acid is DNA.
3. The method of claim 2 , further comprising performing a polymerase chain reaction (PCR) analysis on the nucleic acid sample.
4. The method of claim 1 , wherein the nucleic acid is RNA.
5. The method of claim 4 , further comprising performing a RT-PCR analysis on the nucleic acid sample.
6. The method of claim 5 , wherein the RT-PCR is reverse transcription quantitative real-time PCR (RT-qPCR).
7. The method of claim 1 , wherein the alcohol solution comprises 20% ethanol.
8. The method of claim 1 , further comprising performing a western blot, mass spectrometry protein identification, or ELISA analysis on the protein sample.
9. The method of claim 1 , wherein the filtered samples are stored at ambient temperature for at least two weeks without more than 50% degradation of proteins or nucleic acids present in the filtered samples.
10. The method of claim 1 , wherein the filtered samples are stored at ambient temperature for at least two weeks without more than 25% degradation of proteins or nucleic acids present in the filtered samples.
11. The method of claim 1 , wherein the filtered samples are stored at ambient temperature for at least ten weeks without more than 50% degradation of proteins or nucleic acids present in the filtered samples.
12. The method of claim 1 , further comprising the step of storing the filtered samples at ambient temperature for at least ten weeks without more than 25% degradation of proteins or nucleic acids present in the filtered samples.
13. The method of claim 1 , wherein the alcohol solution comprises 15-25% ethanol.
14. The method of claim 1 , wherein the alcohol solution comprises 5-35% ethanol.
15. The method of claim 1 , wherein the filter is a hydrophilic membrane, and is selected from the group consisting of a 0.22 μm, 0.45 μm and 5.0 μm hydrophilic membrane.
16. The method of claim 15 , wherein the filter is a 0.22 μm hydrophilic membrane.
17. A method for stabilizing nucleic acid and protein samples isolated from a saliva sample, the method comprising:
a) collecting a saliva sample from a human subject;
b) filtering the saliva sample at ambient temperature using a 0.22 μm to 5.0 μm hydrophilic membrane to produce a filtered sample that is free of cells;
c) collecting the filtered sample at ambient temperature in a first and a second receiving device;
d) adding an ethanol solution to the first receiving device at ambient temperature to produce an ethanol-containing filtered sample to form a protein sample, with the proviso that alcohol is not added to the second receiving device to produce an alcohol-free filtered sample to form a nucleic acid sample; and
e) storing the protein sample and the nucleic acid sample for at least 3 days at 25 degrees Celsius.
18. The method of claim 17 , wherein the protein sample is stabilized for at least 2 weeks when stored at 25 degrees Celsius.
19. The method of claim 17 , wherein the nucleic acid sample is stabilized for at least 10 weeks when stored at 25 degrees Celsius.
20. The method of claim 17 , further comprising the step of: f) performing an analysis on the filtered samples collected in the first and second receiving devices comprising one or more of: a protein analysis on the ethanol-containing filtered sample and a nucleic acid analysis on the alcohol-free filtered sample.
21. The method of claim 17 , wherein the ethanol solution comprises 15-25% ethanol.
22. The method of claim 17 , wherein the ethanol solution comprises 5-35% ethanol.
23. The method of claim 17 , wherein the hydrophilic membrane is 0.22 μm.