RT-qPCR array for in vitro screening

Commonly used acronym: qPCR

Scope of the method

Alternative method relates to
  • Animal health
  • Human health
Alternative method is situated in
  • Basic Research
Type of alternative method
  • In vitro - Ex vivo
This method makes use of
  • Human derived cells / tissues / organs
Specify the type of cells/tissues/organs
HepaRG

Description

Method keywords
  • genotoxicity
  • Mutagenicity
  • qPCR array
  • Toxicogenomics
  • HepaRG
  • in vitro
Scientific area keywords
  • in vitro toxicology
Method desription

The RT-qPCR assay is used to identify genotoxic and non-genotoxic compounds. Herefore metabolic-competent human HepaRG cells are exposed to the IC10 value (measured by the MTT test). A microassay is performed to select 84 genes that show the most robust rates of correct classification of the reference compounds into the genotoxic or non-genotoxic group. Each qPCR array also consists of 5 housekeeping genes.

Lab equipment
  • Biosafety cabinet
  • CO2 incubator
  • Bio-rad CFX qPCR instrument
Method status
  • Internally validated
  • Published in peer reviewed journal

Pros, cons & Future potential

Advantages
  • Used genes represent different pathways involved in the DNA damage response
  • All pro-genotoxins included as reference or test compound were correctly classified
Challenges
  • Use of microarray/RNAsequencing platforms
  • Challenging date analysis and interpretation
  • No standalone test, but an important part of an integrated testing strategy
  • Further validation needed
Modifications
  • Use of other cellines
  • Use of other genes

References, associated documents and other information

References

A novel genotoxin-specific qPCR array based on the metabolically competent human HepaRG™ cell line as a rapid and reliable tool for improved in vitro hazard assessment. Gamze Ates,Birgit Mertens, Anja Heymans, Luc Verschaeve, Dimiter Milushev, Philippe Vanparys, Nancy H. C. Roosens, Sigrid C. J. De Keersmaecker, Vera Rogiers, Tatyana Y. Doktorova

Associated documents
Ates2018_Article_ANovelGenotoxin-specificQPCRAr.pdf

Contact person

Jolien Van De Maele

Organisations

Sciensano
Chemical and physical health risks
Risk and health impact assessment
Belgium

Partners

Vrije Universiteit Brussel