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Short Tandem Repeat Analysis in Cell Line and Human Sample Authentication
Short tandem repeat (STR) genotyping is an important
tool in verification of authenticity of human cell lines,
quality control of stored human tissues and fluids, and
assessment of the nature of known mixtures. Many journals and funding agencies now require researchers to authenticate their cell lines prior to their paper or grant being submitted. For biopharmaceutical companies, cell line authentication is used for cell characterization to comply with regulatory guidelines for cell identity records in compliance with cGMP regulations. An STR
analysis workflow that uses capillary electrophoresis
(CE) is a simple, economical method and gold standard
for establishing the identity of human samples.
Speak with a specialist about STR kits and other CE product offerings.
The study of human diseases relies heavily on the analysis of dissociated human cell lines grown in culture. However, cells grown in vitro can be misidentified or become contaminated with other unrelated cell lines. Misidentification of cell lines produces misleading results, confusion, and added costs to research. Journals and funding agencies now require researchers to prove that the cell lines they have used are authentic and have remained so over the course of a study.
Watch this video to find out how STR genotyping can be used to authenticate cell lines.
Sample authentication is carried out when storing or processing biological samples of human origin. Establishing the STR genotype of a sample upon receipt allows one to confirm its identity when the sample is removed from storage or at any point in the workflow.
Watch this video to find out why—and when—sample authentication is important.
Mixed sample analysis (MSA) is the use of an STR chemistry to evaluate the percent mixture between two or three known DNA samples by semi-quantitative endpoint PCR. By increasing the amount of template DNA and decreasing the number of amplification cycles, a balanced profile from both the major and minor contributors can be examined. MSA is performed when the level of a mixture or contamination is critical to a downstream process.
Featured resource
Case study: Matching identities of iPSCs and donors using Identifiler STR profiling kits
The study of human diseases relies heavily on the analysis of dissociated human cell lines grown in culture. However, cells grown in vitro can be misidentified or become contaminated with other unrelated cell lines. Misidentification of cell lines produces misleading results, confusion, and added costs to research. Journals and funding agencies now require researchers to prove that the cell lines they have used are authentic and have remained so over the course of a study.
Watch this video to find out how STR genotyping can be used to authenticate cell lines.
Sample authentication is carried out when storing or processing biological samples of human origin. Establishing the STR genotype of a sample upon receipt allows one to confirm its identity when the sample is removed from storage or at any point in the workflow.
Watch this video to find out why—and when—sample authentication is important.
Mixed sample analysis (MSA) is the use of an STR chemistry to evaluate the percent mixture between two or three known DNA samples by semi-quantitative endpoint PCR. By increasing the amount of template DNA and decreasing the number of amplification cycles, a balanced profile from both the major and minor contributors can be examined. MSA is performed when the level of a mixture or contamination is critical to a downstream process.
Featured resource
Case study: Matching identities of iPSCs and donors using Identifiler STR profiling kits
ANSI/ATCC standards for authentication of human cell lines—standardization of STR profiling
In 2011, a committee of experts convened by ATCC (American Type Cell Culture Collection) introduced the ASN-0002 “Authentication of Human Cell Lines: Standardization of STR Profiling” consensus guidelines. This document, later revised in 2022 (ANSI/ATCC ASN-0002-2022), provides comprehensive guidance on the use of STR analysis for human cell line authentication. ANSI recommends profiling be performed more frequently than every three years and when phenotypic changes are noted in the culture. Additionally, they recommend 13 autosomal STR loci as a standard for authentication: CSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, FGA, TH01, TPOX and vWA.
Source: ANSI/ATCC ASN-0002-2022 Authentication of Human Cell Lines: Standardization of Short Tandem Repeat (STR) Profiling - Revised 2022
Products for PCR-based STR fingerprinting
The Applied Biosystems product portfolio has several different kits for PCR-based STR fingerprinting for use on CE instruments:
- The CLA Identifiler Plus PCR Amplification Kit has been optimized to analyze 16 highly variant human STRs over a wide range of purified gDNA preparations
- The CLA Identifiler Direct PCR Amplification Kit was first developed to analyze the same 16 STR loci, starting from dried blood or buccal spots (for example, on NUCLEIC-CARD devices) or buccal swabs. For the NUCLEIC-CARD device, a 1.2-mm punch from the card is placed directly into a PCR tube or well and amplified without any further purification.
- When extra levels of discrimination are needed, the CLA GlobalFiler PCR Amplification Kit allows 6-dye analysis of 24 loci, 16 of which are included in the Identifiler kits.
GeneMapper Software 6 and the cloud-based microsatellite analysis (MSA) software solutions facilitate analysis of STRs by making use of pre-established allelic ladders and sizing bin sets for the various STR alleles covered by the Identifiler kits.
Figure 1. Workflows for cell line authentication. Two methods are available for cell line authentication. (A) Cells can be spotted onto NUCLEIC-CARD devices, punches of the cards amplified directly using the CLA Identifiler Direct kit, and fragments analyzed on Applied Biosystems CE instruments using GeneMapper Software 6 or MSA cloud application. (B) Alternatively, gDNA can be purified from cell lines, amplified using the CLA Identifiler Plus or CLA GlobalFiler kit, and fragments analyzed by capillary electrophoresis and GeneMapper Software 6 or MSA cloud application.
Product comparison
| Specification | CLA GlobalFiler PCR Amplification Kit | CLA Identifiler Plus PCR Amplification Kit | CLA Identifiler Direct PCR Amplification Kit |
| Markers | 24 (21 autosomal and 3 sex determination markers) | 16 (15 autosomal and amelogenin) | 16 (15 autosomal and amelogenin) |
| Applied Biosystems instrument compatibility | SeqStudio, 3500 series, SeqStudio Flex Series, 3730 series, 3130 series | SeqStudio, 3500 series, SeqStudio Flex Series, 3730 series, 3130 series, and 310 | SeqStudio, 3500 series, SeqStudio Flex Series, 3730 series, 3130 series, and 310 |
| Polymer/array | POP-4, POP-7, 36 cm, and 50 cm | POP-4, POP-7, 36 cm, and 50 cm | POP-4, POP-7, 36 cm, and 50 cm |
| Applied Biosystems software compatibility | GeneMapper software 5 and 6 | ||
| Applied Biosystems thermal cycler compatibility | GeneAmp PCR System 9700 (gold or silver blocks only), Veriti 96-well, and ProFlex PCR System (96-well, 2 x 96-well, and 3 x 32-well) | ||
| Kit size (reactions) | 200 | 200 | 200 |
| Reaction volume (µL) | 25 | 25 | 25 |
| Dye label | 6-dye chemistry (FAM, VIC, NED, TAZ, LIZ, and SID) | 5-dye chemistry (FAM, VIC, NED, PET, and LIZ) | 5-dye chemistry (FAM, VIC, NED, PET, and LIZ) |
| Amplicon size | ≤400 bp; SE33 is under 450 bp | ≤360 bp | ≤360 bp |
| Amplification time | <90 min | 2.5–3 hr | 2.5–3 hr |
| Mini loci–250 bp | 12 full, 3 partial, Y indel, and amelogenin | 10 | 10 |
| Sample input or sample type | 5 µL; total 1 ng but also optimized for 2.5–5 ng | Up to 10 µL; total 1 ng but also optimized for 2.5–5 ng | 1.2 mm punch from a treated paper or 2–3 μL of Applied Biosystems Prep-n-Go Buffer-treated swabs or 1.2-mm punch from an untreated paper |
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