Cell Line Authentication
- Register for iLab: https://uagc.arl.arizona.edu/submit-samples/ilab-operations
- Make a submission on our iLab storefront: https://ua.ilab.agilent.com/sc/4505/university-of-arizona-genetics-core/?tab=about Select the tab “Request Services”, then select "Cell Line Authentication"
- Attach your submission ID (UAGC-NN-########) to your samples and mail it to us, or use our drop off freezers
- If you are on the University of Arizona main campus, you have two options for convenience freezer drop-off. Our Lab is located in the Keating/BIO5 Building Room 106, and there is a freezer just inside the north door to the lab. It is accessible to all from 9-5pm. Alternatively, we have another freezer in Life Sciences South Room 205. Both freezers are checked at least daily.
- Off-campus users can mail their submissions to:
UAGC c/o Liz Cox
1657 E. Helen St. Rm 106
Tucson, AZ 85721-0240
- Regardless of your sample type, all plastic ware should be 1.75 or 2ml snap-cap or screw-capped tubes OR 96-well plate sealed with strip caps. We do not accept Cryotubes, 0.65ml tubes or strips of tubes.
- Cell Pellets – collect ~10^6 cells and put them into a tube filled with 500-700ul DNA lysis buffer. We use the below lysis buffer. Alternatively, a cell pellet without a buffer can be shipped with ice packs. Dry ice is not necessary.
50 mM Tris pH 8.0
50 mM EDTA
25 mM Sucrose
100 mM NaCl
- DNA – send at least 40ul of at least 5ng/ul DNA in either an elution buffer or (not recommended) water. DNA me be extracted by any method that yields purified DNA. If you are quantifying your DNA via nanodrop, please do not dilute the sample prior to shipping.
- Other sample types – we can accept tumor chunks if they are the size of a pea or less. They should ideally be in a DNA lysis buffer. Please contact us ahead of time if you are looking to submit a type of material not covered in this FAQ
- The International Cell Line Authentication Committee (ICLAC) sets a standard by what % of matching STR markers facilitates a line matching to its reference.As cell line may drift with time and passage, and ICLAC set a standard that if two profiles match 80%, the two lines are considered to be related to each other. More specifically, the equation for calculating % match is:
[SHARED ALLELESx2/TOTAL ALLELES in TEST SAMPLE + TOTAL ALLELES in REFERENCE SAMPLE]x100
- You can find more information about ICLAC here: https://iclac.org/resources/match-criteria-worksheet/
- The match threshold of 80% shared alleles was not reached. This may be due to one of the following reasons:
- Your sample is contaminated with a different cell line
- Your sample is not the correct cell line. This is more common when obtaining a line from fellow lab, and less common when ordering from a commercial source that performs STR authentication.
- The line you are working with has already been identified as a different cell line. There are databases of misidentified cell lines which get added to frequently
- The numbers under each heading is the repeat unit at that locus.Either a sample is heterozygous or homozygous for the repeat unit, so there will either be two of the same numbers, or two different numbers.
- X/X and X/Y are the sex chromosomes of the sample, however, it is very common for a line to lose the part of the Y chromosome that is amplified, so a testicular cancer may come back as X/X
- The electropherogram shows us the amplified DNA product which has been fluorescently tagged, and visualized during capillary electrophoresis. The bottom lane is a ladder lane which we use to size the number of repeats.
- The STR assay amplifies non-coding repeat units across the human genome that are used to create a profile, or ‘finger-print’ of an individual.When comparing at least 8 loci, the odds of two non-related people sharing the same profile is 1x10^6. The odds of matching an unrelated individual further decreases the number of loci used to compare profiles.
- The only ability of STR based cell line authentication is to prove that the cells have the same DNA profile as the individual who initially donated the sample. If a single donor has two different cancer types, and both become cell lines, the two lines would be indistinguishable from each other at the DNa level, despite one collected from hepatocarcinoma and the other from a liposarcoma. The assay does not give us any information about tissue type or mutation type.