Validation of reference gene stability for APAP hepatotoxicity studies in different in vitro systems and identification of novel potential toxicity biomarkers.
Liver cell lines and primary hepatocytes are becoming increasingly valuable for in vitro toxicogenomic studies, with RT-qPCR enabling the analysis of gene expression profiles following exposure to potential hepatotoxicants. Supporting the accurate normalisation of RT-qPCR data requires the identification of reference genes which have stable expression during in vitro toxicology studies. Therefore, we performed a comprehensive analysis of reference gene stability in two routinely used cell types, (HepG2 cells and primary rat hepatocytes), and two in vitro culture systems, (2D monolayer and 3D scaffolds). A robust reference gene validation strategy was performed, consisting of geNorm analysis, to test for pair wise variation in gene expression, and statistical analysis using analysis of variance. This strategy identified stable reference genes with respect to acetaminophen treatment and time in HepG2 cells (GAPDH and PPIA), and with respect to acetaminophen treatment and culture condition in primary hepatocytes (18S rRNA and α-tubulin). Following the selection of reference genes, the novel target genes E2F7 and IL-11RA were identified as potential toxicity biomarkers for acetaminophen treatment. We conclude that accurate quantification of gene expression requires the use of a validated normalisation strategy for each species and experimental system employed.
Fox
Bridget C
BC
LGC Limited, Queens Road, Teddington, Middlesex TW11 0LY, United Kingdom.
Devonshire
Alison S
AS
Schutte
Maaike E
ME
Foy
Carole A
CA
Minguez
Jesus
J
Przyborski
Stefan
S
Maltman
Daniel
D
Bokhari
Maria
M
Marshall
Damian
D
D000082:Acetaminophen / Q000633:toxicity*; D018712:Analgesics, Non-Narcotic / Q000633:toxicity; D000818:Animals; D054316:Biomarkers, Pharmacological / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000473:pathology; D002478:Cells, Cultured; D056486:Chemical and Drug Induced Liver Injury / Q000209:etiology* / Q000235:genetics; D050702:E2F7 Transcription Factor / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods; D005786:Gene Expression Regulation / Q000187:drug effects*; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000187:drug effects* / Q000473:pathology; D006801:Humans; D053703:Interleukin-11 Receptor alpha Subunit / Q000235:genetics; D008113:Liver Neoplasms / Q000235:genetics / Q000473:pathology; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013045:Species Specificity
D018712:Analgesics, Non-Narcotic; D054316:Biomarkers, Pharmacological; D050702:E2F7 Transcription Factor; C481054:E2F7 protein, human; D053703:Interleukin-11 Receptor alpha Subunit; D000082:Acetaminophen