Why is it important to get your cell line authenticated?
The validity of research data relies heavily on the identity of the cell line used in testing. According to the American Type Culture Collection (ATCC) Standards Development Organization Workgroup (SDO) ASN-0002, only 33% of researchers test the authenticity of their cell lines. To highlight the problem, the National Institutes of Health (NIH) issued a notice in November of 2007 regarding the authentication of cultured cell lines (Notice: NOT-OD-08-017), which states that “misidentification of cell cultures is a serious problem.”
In addition, standards approved by the American National Standards Institute (ANSI), were issued in 2012 by the ATCC SDO Workgroup to promote authentication of cell lines utilizing STR DNA technology.
Cell line authentication puts your mind at ease about all of the hard work and time you have put into your research.
Can cross-contamination of cell lines or misidentification of cell lines really adversely affect my research data?
Yes! There are researchers that have lost years of work to problems such as cell line cross contamination or misidentification. On August 15, 2010, the authors of a paper originally published on April 15, 2005, retracted their article stating:
“Upon review of the data published in this article, the authors have been unable to reproduce some of the reported spontaneous transformation events and suspect the phenomenon is due to a cross-contamination artifact.”
The 2005 paper, titled “Spontaneous Human Adult Stem Cell Transformation”, included research on human stem cells which were reported to “transform” into cancer-like cells. This 2005 paper was cited over 280 times by other researchers who, like the authors of the paper, were astounded at the possibility that stem cells could be “cancer-like”. This would have implications for ANY researcher performing adult human stem cell studies in research fields such as tissue regeneration and engineering, with fears of treatments leading to tumor growth. The authors of the 2005 paper, unable to reproduce their findings in subsequent years, discovered that there was cross-contamination between the stem cells and a real cancer cell line growing in the same laboratory (HT1080 cells), which upon contamination overgrew the stem cell culture.
Another laboratory published a letter to the Editor in the August 2010 volume of Cancer Research, the same month and year as the retraction paper above, stating “DNA fingerprinting should be compulsory for all experiments involving cell lines. Scientists should verify the cell lines in their possession…” In addition, the laboratory goes on to suggest that “scientific journals should require that all cell lines used in an article [be] verified before publication.”
Do I need to validate my cell line if it was purchased from a repository?
If your cell line was purchased from a repository, an authentication report may have been included with your purchase. Many repositories validate cell lines at regular intervals before selling them, however, cell lines should be continually authenticated after purchase. It is recommended that cell lines be validated every 1-2 months during active growth.
In addition, even if you purchased the cell line from a repository many journals now require cell line authentication as a prerequisite for publication. Contamination and misidentification can happen in any laboratory; it is therefore crucial to constantly check to make sure you are working with the genuine cell line.
A standard Human Cell Line Authentication Report from Genetica DNA Laboratories lists the alleles of the autosomal STR (short tandem repeat) loci plus Amelogenin, (the sex determining marker). Along with the report, we supply you with analytical data printouts (electropherogram). The electropherogram data shows peaks at the different genetic loci, indicating alleles. The electropherogram data is a useful tool when comparing cell line genetic changes ("genetic instability") over time when you have your cell line authenticated as the passage number increases.
Below each peak, there is a set of numbers; the top number is the allele designation (number of short tandem repeats), and the bottom number is the height of the peak measured in RFUs (Relative Fluorescence Units). Because most cell lines are cancer cells, or are tumorigenic, mutations and variations in the DNA are common. This may mean that at some loci, three peaks are visible, or that the peak heights may be unbalanced.
If a mixture of more than one cell line is present there will be more than two peaks at several loci. The peak height value is indicative of the signal strength of that allele, which theoretically should have a direct relationship to the amount of DNA in the sample. Hence, in a mixture of cell lines where some of the peaks at a locus are significantly higher than others, the larger peaks would belong to the cell line that was the major component of the mixture. The smaller peaks in this example would then belong to the minor component of the mixture. Please be aware that a mixture of two or more cell lines can be similar to "genetic instability" in a cell line - where subpopulations of the genuine cell line exist which have a different STR DNA profile due to genetic instability which is inherent to cancerous cell lines - it is therefore crucial that a trained DNA analyst is interpreting the electropherogram data to eliminate a mixture of two or more cell lines as the cause of the additional allele peaks.
What do I do with the information in my Human Cell Line Authentication Report?
For the STR DNA Profile Report (ordered without a comparison report):
A Human Cell Line Authentication Report is necessary in order to compare the STR profile of the cell line that is actually being used for research with the STR profile of the individual from whom the cell line was derived (obtained).
In order to do this, you will need to find a reference to the STR profile your cell line is supposed to have. If you purchased your cell line from a repository, they may have given you this information with the cell line upon purchase. Also, you may be able to find the profile in a published paper.
One suggestion on how to find your cell line’s STR profile is to go to www.atcc.org. Here you can search by the cell line’s name or ATCC number, if available. Once the search results come up, find the cell line you are interested in and click on it. This will bring up a page with a description of your cell line, including the DNA profile at nine STR sites. All of these nine STR sites are part of the DNA profile you’ve received from Genetica DNA Laboratories on your report.
From here you can compare the original cell line’s profile, with the profile on the Authentication test report of the cell line sample submitted to Genetica. If they are the same, it means that you are working with the authentic cell line. If they are different, you have experienced a problem of some kind; this could mean misidentification, mislabeling, or contamination by a different cell line. A mixture of two or more cell lines would indicate that a second cell line has been introduced to your working sample, contaminating it.
Another suggestion is to use an online verification tool to confirm the identity of the human cell line. One such available database can be found at www.dsmz.de. Click the link to “Human and Animal Cell Lines.” Then, click the link along the side of the page, “Online STR Analysis.” Here you will be asked to register (it’s free). Next, you type in the DNA profile at the nine (9) sites listed (all of which are included in your report), as the online example shows. The database shows which cell lines are the closest match to the profile you typed in. Values close to one (1) are indicative of a close match to the query. If the tested cell line has not been contaminated or misidentified, the name should appear high on the list with a value close to one (1). If the tested cell line has been contaminated or misidentified, the actual name of the cell line will not be on the close match list, and instead the contaminating cell line will be listed.
If the tested cell line has been contaminated or misidentified, additional authentication testing should be done on earlier passages to identify where the problem started, or a new sample from a repository should be obtained. Any research or experiments done with a contaminated or misidentified cell line may have to be performed again before publishing or moving forward with your research.
A cell line authentication STR Comparative Analysis Report from Genetica DNA Laboratories is a report containing all the information you need to determine if your submitted cell line is the genuine cell line. The Comparative Analysis Report lists the expected DNA profile and the DNA profile of the cell line you submitted to Genetica for testing. In addition, the percent identity and percent match calculations of your submitted cell line compared to the genuine reference cell line is calculated for you. Sub-lines of the same cell line may not have identical STR profiles (100% identity is rare). According to ATCC, a threshold of 75% identity and above is acceptable to consider your submitted cell line as genuine. With a % identity between 50-75%, the cell line should be regarded with suspicion. Below 50% identity, the submitted cell line is considered different from the genuine cell line [Nature Reviews Cancer 10:441, 2010].
Why should I trust Genetica DNA Laboratories with my cell line?
Genetica DNA Laboratories - a LabCorp Specialty Testing Group, has been in business for over 20 years. Genetica DNA Laboratories provides quality DNA testing including parentage, family relationship, and identity testing, as well as, cell line authentication testing. Our laboratory is CLIA certified as well as being accredited by the College of American Pathologists (CAP), Accredited to ISO/IEC 17025) by ANSI-ASQ National Accreditation Board/FQS, the American Association of Blood Banks (AABB), and the New York State Department Of Health (NYSDOH). Furthermore, Genetica offers top notch customer service. Please feel free to call us anytime with questions or for a consultation!
My lab is not in the United States, do you accept samples for cell line authentication from other countries?
Absolutely! We currently have clients from around the world. The best option for sending samples overseas is to extract and quantitate the DNA from your cell line sample(s) in your own laboratory. The extracted DNA from your cell line sample(s) do NOT need to be on ice; shipping at ambient temperature is sufficient and causes no problems with the cell line authentication testing process. Sending a bulky dry ice package internationally could be expensive and potential extended shipping delays could damage the sample(s). If you prefer to send cell pellets (frozen or dried) - please call Genetica for the best option for your specific needs. We are more than happy to assist!
Genetica® DNA Laboratories
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