How To

Testing FAQ’s

Are there any recommended pipetting techniques?

​The key to accuracy in pipetting is a consistent technique.  Consistent speed and smoothness while pipetting is necessary.  Avoid any sudden motions when drawing or dispensing fluids.

Always pre-wet the pipette tips – see; Pre-wetting the pipette tip(s) below. (Always change pipette tips between each standard, control and sample!)

Pre-wetting the pipette tip(s) will result in more accurate and consistent pipetting of solutions which is very important for pipetting duplicates onto an immunoassay plate.  Pre-wetting tips also applies to multichannel pipettes.

a.Firmly place the correct size pipette tip onto the end of the pipetter.

b.Depress the plunger button at the top of the pipetter with your thumb until you feel the first stop point.

c.Immerse the pipette tip under the surface of the liquid to be dispensed to a depth of 2-5 cm, staying clear of the container walls and bottom.  Release the plunger button slowly to draw liquid up into the tip.

d.Pause with the tip in the liquid for one or two seconds after aspirating.  The liquid in the tip “bounces” slightly when the plunger is released.  A slow, consistent pause helps minimize errors due to this effect.

e.Withdraw the tip from the liquid & dispense back into the container by gently pressing the plunger all the way down.  This “pre-wets” the tip.

What should I do if my saliva samples are viscous?

The key to accuracy in pipetting is a consistent technique.

Consistent speed and smoothness while pipetting is necessary. Avoid any sudden motions when drawing or dispensing fluids.

Always pre-wet the pipette tips – see; Pre-wetting the pipette tip(s) below. (Always change pipette tips between each standard, control, and sample!)

Pre-wetting the pipette tip(s) will result in more accurate and consistent pipetting of solutions which is very important for pipetting duplicates onto an immunoassay plate. Pre-wetting tips also applies to multichannel pipettes.

a. Firmly place the correct size pipette tip onto the end of the pipetter.

b. Depress the plunger button at the top of the pipetter with your thumb until you feel the first stop point.

c. Immerse the pipette tip under the surface of the liquid to be dispensed to a depth of 2-5 cm, staying clear of the container walls and bottom. Release the plunger button slowly to draw liquid up into the tip.

d. Pause with the tip in the liquid for one or two seconds after aspirating. The liquid in the tip “bounces” slightly when the plunger is released. A slow, consistent pause helps minimize errors due to this effect.

e. Withdraw the tip from the liquid & dispense back into the container by gently pressing the plunger all the way down. This “pre-wets” the tip.

How should I label my samples to send to Salimetrics for testing?

​For samples being sent to Salimetrics, label the exterior of the tube using pre-printed, bar-coded labels provided, or write the ID number with a waterproof pen. Further details can be found in the Saliva Collection Handbook on page 4.

How long can my sample stay on the plate before adding conjugate/substrate?

​To ensure highest quality assay results, pipetting of samples and reagents must be done as quickly as possible (without interruption) across the plate. Ideally, the process should be completed within 20 minutes or less.

Is it cost effective to send samples to Salimetrics for testing or should we do the testing ourselves using Salimetrics kits?

​Utilizing a high quality lab will improve the quality of your data. If your immunoassay lab has highly experienced technicians, the latest equipment, and is up-to-date on current saliva collection, handling, storage, and testing protocols, it can cost less to test small studies yourself. For large projects, the SalivaLab is often the most cost-effective option when high-quality data is desired. If you would like to learn how to get high-quality data in your lab, we would welcome you at “Spit Camp” to teach you how to do this. Many who come to Spit Camp decide very quickly if it’s worth it to send samples to Salimetrics. There are other options as well. Salimetrics has a network of laboratories we call “Centers of Excellence (COE) Labs”. We monitor their performance on a regular basis. If there is a COE in your region, this is a very good alternative to consider so you will get guaranteed, reliable results using our laboratory products.

How do I ship samples to Salimetrics for testing?

​Complete instructions on how to send your samples to Salimetrics can be found under the Send Samples link at the SalivaLab.

Why do I have to freeze and centrifuge saliva samples before testing them?

​Whole saliva contains glycoproteins know as mucins, which form mucus when dissolved. This thick and sticky substance can cause problems in transferring small volumes by pipette. Freezing saliva samples will precipitate mucins. Centrifuging removes mucins and other particulate matter which may interfere with antibody binding, leading to falsely elevated results.

Do I need to keep samples cold for saliva testing?

​Salimetrics only accepts samples that are shipped on dry ice to the SalivaLab.

Can I send/ship samples from outside the United States to Salimetrics for assay?

Yes. To ship samples to Salimetrics from outside the United States:• Contact Salimetrics for specific instructions regarding documentation that must accompany your samples. A deviation from the proper procedure will cause a delay in getting samples through United States Customs.• Transit times may be more than one day. Please take this into account when packaging your samples.• Shipping regulations may differ outside of the United States. Please investigate this before shipping. Visit the SalivaLab site at; http://www.salimetrics.com/submit-samplesfor more information.

DNA FAQ’s

Can I use saliva collected for DNA with the Oragene device to test for other analytes?

​No, the fluid in the Oragene device will dilute the saliva by an unknown amount and also has the potential to interfere with EIA, thus impeding the accurate measurement of analytes.

Can I use saliva collected for DNA to test for other analytes?

​Yes, saliva collected for DNA analysis using SalivaBio or Salimetrics collection devices can be used for other analytes as well. DNA in whole saliva is obtained from the cell pellet after centrifugation.  The supernatant can be used for testing other analytes.  DNA is collected from the actual SalivaBio swabs after centrifugation, so the saliva in the swab storage tube can be used testing other analytes.  Salimetrics recommends collecting the DNA material before testing for other analytes but it still possible to get the DNA sample after testing for other analytes provided that care is taken to prevent cross contamination during the testing phase. See this publication for more information;

Zsofia Nemoda, Maria Horvat-Gordon, Christine K Fortunato, Emilie K Beltzer, Jessica L Scholl and Douglas A Granger;Assessing genetic polymorphisms using DNA extracted from cells present in saliva samples;BMC Medical Research Methodology 2011, 11:170 doi:10.1186/1471-2288-11-170.

How do I use your saliva collection devices to collect samples for DNA testing?

​Information on this topic was published online in the following paper:

Nemoda, Z.., Horvat-Gordon, M., Fortunato, C. K., Beltzer, E., Scholl, J. L., Granger, D. A. (2011). Assessing genetic polymorphisms using DNA extracted from cells present in saliva samples.  BMC Medical Research Methodology, 11, 170. PMID 22182470

Handling and Storage FAQ’s

Is it ok to have saliva frozen in the home and mailed overnight to a central collection location?

​For home saliva collection, if shipping on dry ice is not possible/practical, then they should be frozen and shipped frozen using 3 ice paks in a u-line insulated cooler for 36 hour delivery.  We advise including a temperature tracking device in your shippers. Salimetrics recommends FedEx overnight shipping.

Can saliva samples be shipped by individuals in the regular mail?

​Choices of how samples will be shipped depends on several variables including budget constraints, shipping distance, domestic or international source of origin, and ability to control the cold chain. Ideally, samples once frozen after collection would stay frozen. Salimetrics website has advise about how to pack and ship samples to maintain the cold chain. Generally speaking, samples coming from individuals should be sent to a project coordination site locally, batched and then shipped on dry ice to Salimetrics. We advise including a temperature tracking device in your shippers, and these devices can be obtained via Salimetrics. Salimetrics only accepts samples that are shipped on dry ice to the Salimetrics SalivaLab.

What is Salimetrics recommendation for saliva storage? Whole saliva (passive drool)?

​Saliva handling and storage after collection is critical to the process of generating high quality results. Some analytes are unstable when handled at room temperature. Peptides (e.g., oxytocin) are so unstable that we recommend the samples be flash frozen within minutes of collection. Most analytes in saliva do not require flash freezing to maintain sample integrity. One of the major differences between saliva and traditional biospecimens is bacteria load.  Saliva has many different types of bacteria, some types are present in very high levels, and the types and levels vary from person to person. To protect unstable analytes and to prevent bacterial growth, we advise that all samples should be frozen at -20 C or below as soon as possible. If freezing is not practical, then it is appropriate to hold samples temporarily (for hours) at 4ºC. Samples stored for more than 4-5 months should be frozen at -40ºC or below.

You can find more detailed information in the Salimetrics’Saliva Collection Handbookon page 5.

How long can saliva for each analyte be stored frozen at -60C or below?

Once frozen at – 40 C or below, the stability analytes in saliva is no different than the long term stability of analytes in other traditional biological specimens. Theoretically, storage at -40 C or below is the best option and most economic option for the storage of saliva samples in biorepositories.

How do I aliquot samples?

​In this video we demonstrate how to split saliva samples via aliquoting. We demonstrate proper technique and also review workspace prep and sample organization.