About Cell Line Validation
Between 18% and 36% of cell lines are either misidentified or cross-contaminated (list of misidentified cell lines). A recent notice from NIH requires cell line validation for grant applications to be considered of the highest quality. Journals such as Science, Nature and PNAS also require cell line validation for publication. Cell lines that have been extensively characterized at the DNA, RNA and protein levels will allow investigators to choose the correct cell line for their research. Pre-characterized cell lines will decrease the cost to researchers since this will eliminate repeat analysis. Thus, cell line validation is a critical issue for both scientific publications and grant applications.
*MD Anderson's Cell Line Authentication Policy
MD Anderson's policy (ACA#1044) now requires all researchers to validate their cell lines at least once per year (Our Core highly suggests to validate cell lines every 6 months).
*SNP_ID Assay for Sample Validation (Replacing STR Service)
The SNP_ID is a highly multiplexed assay that provides a cost-effective method for accurate and comprehensive sample comparison. It is suitable for DNA from cell lines, tissue and blood samples. The assay has 44 SNPs selected with 45-55% heterozygosity across six major HapMart populations, 3 markers for gender identification, and 5 markers for sample quantification, designed into a single multiplexed assay. It can be used for cell line validation, as well as for tumor-to-normal matching. One of the main advantages of the SNP_ID versus STR for sample validation and comparison is that it works with degraded DNA, as amplicons are smaller, as compared to STR where some loci might require hundreds of bp. Additionally, due to its highly multiplexed nature, SNP_ID assay is less expensive to run than STR. This new assay will be run using our sequenom instrument.
About Mutation Detection (Sequenom)
The CCLC offers a hotspot mutational analysis that is performed using the Sequenom MassARRAY using the iPLEX technology. This technology allows for parallel high-throughput screening while using minimal DNA obtained from fresh frozen or FFPE specimens. The analytical sensitivity of the assay is higher than conventional Sanger sequencing (Limit of detection-LOD: 10%–20%) and similar to pyrosequencing (LOD: 5%–10%). Mutations are screened by using amplification through polymerase chain reaction (PCR) and single-base primer extension where the wild type or mutated base was identified by mass spectrometry. For data analysis, we used Sequenom Typer Software for visual inspection and interpretation of mass spectra. Reactions where the mutant peak represented more than 10% of the wild type peak were scored as positive. The data analysis was performed using MassArray TYPER 4.0 genotyping software (Sequenom) where the SNP calls were divided in 3 groups: conservative, moderate and aggressive calls, depending on the level of confidence. The Sequenom panel used here was previously designed by the Characterized Cell Line Core (Core Shared Resources – CCSG) at MD Anderson Cancer Center with the aim of detecting somatic DNA alterations in cancer samples. A total of 179 point mutations in 33 genes frequently mutated in solid tumors were analyzed. (See Panels A & B available)
About Mycoplasma Detection
Mycoplasma is a bacterial contaminant commonly found in cell culture. It is easy to spread from culture to culture, and unlike most other bacterial or fungal contaminants, mycoplasma contamination cannot be easily detected without further testing. Mycoplasma are bacteria that do not contain a traditional cell membrane and can alter the metabolic characteristics of cells in culture negatively affecting experimental results. We provide this mycoplasma determination service using the Lonza MycoAlert Kit.
Purchase Cell Lines
The CCLC has a List of Cell Lines that have been validated and tested for mycoplasma contamination that are available for purchase. Please see the Services page for pricing and instructions on requesting cell lines.
Core Grant Citation
This facility is funded by NCI # CA016672. Publications should cite the Core grant in the acknowledgment section:
“STR DNA fingerprinting was done by the CCSG-funded Characterized Cell Line Core, NCI # CA016672.”
Two copies of the publication acknowledging the CCLC grant should also be submitted to the facility at Unit 1058.
Education & References
- Lacroix M. Persistent use of "false" cell lines. International Journal of Cancer, 2008. 122(1):1-4.
- Bravo NR, Gottesman M. Notice Regarding Authentication of Cultured Cell Lines. 2007 [cited 2008 1/28/08].
- Rae JM, et al. MDA-MB-435 cells are derived from M14 melanoma cells--a loss for breast cancer, but a boon for melanoma research. Breast Cancer Research & Treatment, 2007. 104(1):13-9.
- Chatterjee R. Cell biology. Cases of mistaken identity. Science, 2007. 315(5814):928-31.
- Drexler HG, et al. False leukemia-lymphoma cell lines: an update on over 500 cell lines. Leukemia, 2003. 17(2):416-26.
- MacLeod RA, et al. Widespread intraspecies cross-contamination of human tumor cell lines arising at source. International Journal of Cancer, 1999. 83(4):555-63.