STR Profiling and Mycoplasma Detection testing
Misidentified cell lines could lead to invalidation of data, lost time, money and effort. In fact, studies have estimated that up to 20% of published papers could be invalid due to misidentified or cross-contaminated cell lines.
Since 2016, many journals and funding agencies have started requiring authentication of human cell lines prior to a paper or grant submission.
Research excellence in tissue culture practices require cultured cells to be maintained free of mycoplasma infections. QIMR Berghofer provides a service for the detection of viable mycoplasma in tissue culture experiments. The luminescence assay that was selected only detects viable/live mycoplasma species. Mycoplasma detection assays are a selective biochemical test to detect mycoplasma contamination in cell cultures. They exploit the activity of mycoplasma enzymes; these enzymes are found in the vast majority of about 200 mycoplasma species but are not present in eukaryotic cells.
Human cell line authentication relies on the analysis of Short Tandem Repeats (STRs) that are detected through the use of capillary sequencers. STR's are repetitive sequence elements 2-7 base pairs long that are located throughout the human genome. These sequences are polymorphic – the specific pattern of a locus can be repeated any number of times. In 2011, the American National Standards Institute (ANSI) and the American Type Culture Collection (ATCC) published consensus guidelines for best practices in cell line authentication using STR genotyping. First, STRs are amplified by PCR using primers outside the repeat sequence. The resulting amplicons are separated using capillary sequencing to determine the number of repeats in the sample. The resulting profile is then compared with a reference profile to verify the origin of the cell line. The statistical power of discrimination increases as you amplify more loci, due to the repeat variation among humans and human cell lines derived from individuals.
The ANSI/ATCC Guidelines indicate that it is unlikely that any single cell line would have more than two alleles in three or more loci. Multiple alleles at multiple loci are another indication of cross-contamination with multiple cell lines in the one culture.
Services
Human cell line authentication uses Short Tandem Repeats (STR) genotyping to validate cell line authenticity. A minimum of 10 markers are used to identify a human cell line, all allelic reports conform with the ATCC ASN-0002 standard. A small amount of good quality genomic DNA is needed (~50ng) to complete the assay for authentication. If more alleles are required, we can accommodate this request by using a different amplification kit. Please ask about options.
Mycoplasma detection testing is achieved with minimal impacts to the tissue culture process and is achieved by using a bioluminescence assay to detect viable mycoplasma species. We provide a service for the detection of viable mycoplasma in spent tissue culture media. Detection of mycoplasma in cell cultures is performed as follows: Viable mycoplasma in a test sample (cell culture supernatant) are lysed and the released mycoplasma enzymes react with the MycoAlert® Substrate, catalysing the conversion of ADP to ATP. The ATP is then transferred into a light signal via the luciferase enzyme in the MycoAlert® Reagent. By measuring the level of ATP in a sample both before (read A) and after the addition of the MycoAlert® Substrate (read B), a ratio can be determined that indicates the presence or absence of mycoplasma.
The luminescence assay only detects viable/live mycoplasma with only small amounts of spent culture media needed. Approximately 1mL is required with the assay detecting as low as 50 CFUs of viable mycoplasma.
Please email: analytical.facility@qimrb.edu.au
Facility Staff
Paul Collins
Chaitanya Dasari
Tu Parsons
Instruments and Platforms
ABI SeqStudio Flex 8 Genetic Analyser 2
Synergy Neo – Fluorometric, Luminescence and UV/Vis assays.