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Northwestern University Feinberg School of Medicine
Center for Genetic Medicine
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Human Cell Line Authentication through STR Profiling

Projects based on the use of cell line(s) may have a need to validate the cell line(s) being used, as their identity could have changed over time due to natural genomic alterations or contamination. For projects that start anew, the importance of authenticating the cell line(s) to be used, potentially for years to come, is apparent. The risks of using unauthenticated cell lines include adding misinformation to scientific literature, waste of funding and resources, and irreproducibility of results.

Cell line authentication (CLA) is an essential aspect of addressing the increasing concern for scientific rigor and reproducibility. This has led to the establishment of requirements for proof of cell line identity and purity by funding agencies (including NIH) and journals (including Nature journals, whole list). The #authenticate campaign launched by Global Biological Standards Institute (GBSI) put the focus on the importance of CLA and has led to the establishment of Cell Authentication Alliance with cosignatories including a number of nonprofit funding agencies.   

There is an array of methods for testing cell line vis-à-vis species, chromosomal structural changes, spontaneous mutations/genetic drift, contamination with adventitious agents, and interspecies/intraspecies contamination. These methods include DNA-based species barcoding, karyotyping, profiling of STRs (Short Tandem Repeats), genotyping of SNPs, PCR amplification using species-specific primers, and whole-genome sequencing. While WGS will eventually provide thorough evaluation of a cell line, STR profiling is by far the most developed and cost-effective approach for authenticating human cell lines (details on STR profiling). Similar to forensic DNA identification, assaying a relatively small number of STR loci provides enough power to discriminate different human cell lines.

NUSeq uses a commercially available kit (AmpFLSTR® Identifiler® Plus PCR Amplification Kit) for STR profiling. This is the same kit used by forensic scientists for casework. This kit is a multiplex STR assay allowing amplification of 15 tetranucleotide repeat loci and the Amelogenin marker (for gender determination) in a single PCR amplification.

Human Cell Line Authentication Pricing

Human cell line authentication costs are listed on the Core Pricing page.

Human Cell Line Authentication

 Sample Preparation

Please fill out the submission form (Excel).

Dilute extracted cell DNA samples in low TE buffer (only with 0.1 mm EDTA) as too much EDTA is inhibitory to PCR. If possible, elute the DNA sample with low TE buffer during DNA extraction and please contact the technical support of the company of your DNA extraction kit to confirm the feasibility.

Please put the DNA concentration, concentration measurement method (such as Nanodrop, Qubit) and 260/280 ratio (if known; this information is important for us to determine your samples’ quality) in the order form.

Sample ID should be no more than eight letters/numbers (no blank or special characters). The sample ID in you order form must match the ID on the top of the sample tub.

Minimum volume of each genomic DNA sample is 20uL, and the required concentration is 10ng/ul.

Samples can be shipped at ambient temperature.

 Sample Submission And How To Order

If needed, project consultation is provided free-of-charge. Cell Line Authentication services can be requested through NUcore. If you need to set up a NUCore account to place an order please visit our Access NUSEQ Services Page

 

There are several ways you may send your samples to the core:

If you are from Northwestern and are on the Chicago campus:

Ban bring your samples directly to the facility's lab location. Fresh samples brought directly to the core should be stored in the DNA Extraction room refrigerator. Please drop off samples directly to a technician at the Core if they need to be stored at -80°C.

If you are from Northwestern and are on the Evanston campus:

Drop your samples off at one of the two courier locations in the Hogan Building, 2205 Tech Drive: Hogan 2-100, BMBCB administrative office, or Hogan 4-150. The couriers pick up the samples every morning and we receive them in the afternoon.

If you are not on the Northwestern campus:

Please send your samples through an independent carrier such as FedEdx or UPS to the address listed under Contact Us. Prior to shipping, please contact a technician to schedule delivery. At the time of shipping, please send an email notification with your express mail tracking number. This will allow us to watch for your shipment. We prefer that samples are sent overnight on Monday or Tuesday so that we can guarantee someone is here to accept them. We will notify you when your shipment is received.

Please fill out the submission form (Excel).

 How to Use Our Data

To determine whether the test cells are related to the reference cells in the STR database, enter the STR marker information we provide in the report (registration required).

For detail, please read "View our brief tutorial before starting.

As shown below, Information on the STR markers of your cells can be found in the allele 1, 2 or 3 columns in the report sent to you.  

The ATCC website will automatically provide the percent match of your test cell lines with the reference cell line in the database. However, we recommend that you calculate it manually to double check, as the result provided by the ATCC database is not always correct. Please check "how to calculate percent match" below for more details. 

The percent match determines whether cell lines are related or not. Cell lines matching at no less than 80% of alleles across 8 major loci are considered to be related and can be authenticated. Although there are several methods to calculate the percent match, the life technology scientist recommends using the method shown in ASN-0002: Authentication of Human Cell Lines: Standardization of STR Profiling

Percent Match The number of SHARED ALLELES of two cell lines X (%) / The total number of alleles in questioned profile (test cells line)

The percent match is based on comparing only the eight core STR markers plus amelogenin of the questioned profile (test cell lines) with those markers in reference cell lines in the database.  

The following table hows how to calculate percent match.  

Designation 

U-87 MGGlioblastoma-AstrocytomaHuman 

question( test) cell line 

D5S818 

11,12 

11, 12 

D13S317 

8,11 

8, 11 

D7S820 

8,9 

8,9 

D16S539 

12 

11 

vWA 

15,17 

15,17 

TH01 

9.3 

9.3 

AMEL 

X,Y 

X

TPOX 

CSF1PO 

10,11 

10,11 

Percent  Match 

92.8% 

 

As shown above, there are 13 shared alleles between the questioned profile (test cell line) and the reference cell lines in the database. There are 14 alleles in total in the questioned cell lines. Therefore, the percent match of the test cell line and the reference cell line, U-87, is 92.8 percent. Please note that the homozygous allele is counted as one allele. To protect the privacy of the cell donor, the database only provides eight core STR markers plus amelogenin and does not provide information for the rest of seven STR markers, which were shown in our report. However, the information from these eight core STR markers are enough to authenticate a cell line for research and publication purposes. The information for the rest of SRT markers only serves as internal reference.   

Visit these sites for more information:

 Turnaround Time

It may take us two to three weeks to complete the project. Please contact us for more information.

Frequently Asked Questions

 How much does it cost?

Please refer to our Core Pricing page.

 Besides ATCC, what other STR databases are available to search STR profile against?

DSMZ (Germany), JCRB (Japan), and RIKEN (Japan) maintain public databases of authenticated human cell lines including their STR profiles. The DSMZ site provides a search function for all these databases to identify matches. The NCBI’s BioSample database also stores STR profiles of publicly catalogued human cell lines, as well as currently known misidentified cell lines collected by the International Cell Line Authentication Committee (ICLAC).

 How often should CLA be conducted?

There is still no consensus as to how often CLA should be conducted. There is a growing number of publications addressing this issue though (e.g., a BioTechniques paper).

 How about authentication of nonhuman cell lines?

STR profiling is expected to work similarly for nonhuman CLA. But so far there is no established assay to use. To develop new assays for nonhuman cells following the established ANSI/ATCC standard for human cell lines, a community effort is required for at least the following: identification of SRT loci for reliable, consistent intraspecies discrimination across different labs, establishment of raw data metrics for determination of data quality, determination of data analytics for STR marker matching, including what level of matching is accepted as a “match.” There is some progress being made toward authentication of nonhuman cell lines. For example, a mouse cell line authentication consortium has been formed from a partnership between ATCC and the National Institute of Standards and Technology (NIST).

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