Protect your research from contaminated or misidentified cell lines.

eCould Be At Risk...Your Cell Lin s

INTRODUCTION

Human cultured cell lines are used in many biomedical research and clinical applications, including cancer research, drug discovery, genetics and biobanking. However, misidentification of cell lines continues to plague research, despite prominent scientists calling for authentication.

Reference: Capes-Davis A., Reid Y.A., Kline M.C., Storts D.R., Strauss E., Dirks W.G., Drexler H.G., MacLeod R.A., Sykes G., Kohara A., Nakamura Y., Elmore E., Nims R.W., Alston-Roberts C., Barallon R., Los G.V., Nardone R.M., Price P.J., Steuer A., Thomson J., Masters J.R., Kerrigan L. (2013) Match criteria for human cell line authentication: where do we draw the line? Int. J. Cancer 132, 2510–19.

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Why are researchers concerned?

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For more information, see: www.promega.com/CLA

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Misidentified or contaminated cell lines lead to invalidation of data as well as lost time, money and effort.

Authentication of human cell lines is now required or strongly encouraged by many journals and funding agencies.

ANSI Standard ASN-0002-2011 recommends the use of STRs for human cell line authentication.

STR testing is recommended when receiving a cell line from an unreliable source, after ten passages, when preparing a cell bank, and when in doubt of its origin or identity.

Researchers are concerned, because:

Cell line contamination is EXPENSIVE

18 to 36%

of cell lines are misidentified

CAT

When should you authenticate a

cell line?

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When establishing or acquiring a new cell line.

Within the first week of passaging a new culture.

When starting a new series of experiments.

When routinely passaging cell lines.

When observing inconsistent cell behavior or unexpected results.

When preparing to publish.

When freezing cell stocks to verify purity.

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Cell Line Authentication is achieved through the use of Short Tandem Repeats (STRs). STRs are repetitive sequence elements 3-7 base pairs long scattered throughout the human genome. By amplifying and analyzing these polymorphic loci, then comparing the resulting STR profile to that of a reference sample, the origin of biological samples such as cells or tissues can be identified and verified. The more loci that are amplified, the higher the statistical power of discrimination due to the repeat variation among humans and the human cell lines derived from individuals.

How does it work?

STR ANALYSIS OF CELL LINESThe recommended steps (see illustration below):

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Send cells or extracted DNA to an experienced STR profile testing service.

Compare the resulting STR profile to a reference sample and other known cell line STR profiles.

Check the cell line name against the database of misidentified cell lines.

DNA EXTRACTION

MULTIPLEX PCR

DATA ANALYSIS

DATABASE COMPARISON

STR ALLELE SEPARATION & SIZING

Reference: Database of Cross-contaminated or Misidentified Cell Lines - http://iclac.org/databases/cross-contaminations/

The American Tissue Culture Collection (ATCC) Standards Development Organization Workgroup published ASN-0002-2011, which recommends the use of at least eight STR loci (TH01, TPOX, vWA, CSF1PO, D16S539, D7S820, D13S317 and D5S818) plus Amelogenin for gender identification for human cell line authentication. The probability of two human cell lines with identical STR profiles using the eight core STR markers is 2 × 109.

What is ASN-0002-2011?

Reference: ANSI/ATCC ASN-0002-2011. Authentication of human cell lines: Standardization of STR profiling. ANSI eStandards Store, 2012.

PUBLISHED!ASN-0002-2011

Plan your next study

Look to the future

Get on with your research

STR products from Promega allow you to achieve confidence in the identity of your cell lines.

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GenePrint® 10 System

The GenePrint® 10 System is a complete system with all reagents required for successful identification and authentication of human cell lines and detection of cell line cross-contaminations.

• Co-amplification and detection of nine STR loci, plus Amelogenin

• Direct amplification of DNA samples from storage card punches

• Amplification of up to 10ng of DNA template

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A = Amelogenin

D3S1358 D1S1656 D2S441 D10S1248 D13S317 Penta E

D16S539 D18S51 D2S1338 CSF1PO Penta D

TH01 vWA D21S11 D7S820 D5S818 TPOX

D8S1179 D12S391 D19S433 SE33 D22S1045

A

7426

MD100 200 300 400

Fluorescein:

JOE:

TMR:

CXR:Size (bp)

TH01 D21S11

D5S818

vWA TPOXA

A = Amelogenin

D13S317 D7S820 D16S539 CSF1PO

60 100 200 300 400 500 1091

0MC

A = Amelogenin

A = Amelogenin

D3S1358 D1S1656 D2S441 D10S1248 D13S317 Penta E

D16S539 D18S51 D2S1338 CSF1PO Penta D

TH01 vWA D21S11 D7S820 D5S818 TPOX

D8S1179 D12S391 D19S433 FGA D22S1045

7426

ME

100 200 300 400

Fluorescein:

JOE:

TMR:

CXR:Size (bp)

TH01 D21S11

D5S818

vWA TPOXA

D13S317 D7S820 D16S539 CSF1PO

A

DYS391

The GenePrint® 10 System allow co-amplification and three-color detection of 10 loci, including all ASN-0022-2011 (TH01, TPOx, vWA, CSF1Po, D16S539,

D7S820, D1S317, D5818, D21S11) and Amelogenin.

GenePrint® 24 System

The GenePrint® 24 System is a 24-locus multiplex system designed to generate a multi-locus human DNA profile from a variety of human- derived biological sources.

• GenePrint® 24 is optimized for mixed sample analyses

• Small kit size, 100 reactions for 12.5ul reaction volume

• Cycling conditions for 2.5 and 5 ng of DNA template

• Protocols for POP-7 and 50 cm capillary arrays

This system allows co-amplification of Amelogenin, D3S1358, D1S1656, D2S441, D10S1248, D135317, Penta E, D16S539, D18S51, D2S1338, CSF1PO, Penta D, TH01, vWA, D21S11, D7S820, D5S818, TPOX, DYS391, D8S1179, D12S391, D19S433, FGA and D22S1045.

60 100 200 300 400 500 1091

0MB

A = Amelogenin

D3S1358 D1S1656 D2S441 D10S1248 D13S317 Penta E

D16S539 D18S51 D2S1338 CSF1PO Penta D

TH01 vWA D21S11 D7S820 D5S818 TPOX

D8S1179 D12S391 D19S433 SE33 D22S1045

A

7426

MD100 200 300 400

Fluorescein:

JOE:

TMR:

CXR:Size (bp)

TH01 D21S11

D5S818

vWA TPOXA

A = Amelogenin

D13S317 D7S820 D16S539 CSF1PO

60 100 200 300 400 500 1091

0MC

A = Amelogenin

A = Amelogenin

D3S1358 D1S1656 D2S441 D10S1248 D13S317 Penta E

D16S539 D18S51 D2S1338 CSF1PO Penta D

TH01 vWA D21S11 D7S820 D5S818 TPOX

D8S1179 D12S391 D19S433 FGA D22S1045

7426

ME

100 200 300 400

Fluorescein:

JOE:

TMR:

CXR:Size (bp)

TH01 D21S11

D5S818

vWA TPOXA

D13S317 D7S820 D16S539 CSF1PO

A

DYS391

Biology – Animal

Journal of Molecular Biology

Journal of the National Cancer Institute

Molecular Vision

Nature Publishing Group

Neuro-Oncology

Placenta

These publications require some level of cell line authentication before accepting research articles:

Journals Requiring Cell Line Authentication

AACR Journals

Endocrine Society Journals

Society for Endocrinology Journals

Carcinogenesis

Cell Biochemistry and Biophysics

Cell Biology International

International Journal of Cancer

In Vitro Cellular & Developmental

Experienced STR Profile Testing Service Providers in North America

ATCC

Biopolymer/Genomics Core Facility

Cell Line Genetics

Dana-Farber Cancer Institute

DDC Medical

Duke University

Genetica – LabCorp

Genewiz

GenoSeq

Hospital for Sick Children – TCAG

IDEXX RADIL

Johns Hopkins University

Laragen

Michigan State University

University of Arizona Genetics Core

University of Illinois at Chicago

Wayne State University Genomics Core

DiagCor

The Garvan Institute

AGRF

MHTP Medical Genomics Facility

To learn more about the information presented here, please go to: www.promega.com/CLA