title: Doxorubicin Treatment of Cancer Cells Impairs Reverse Transcription and Affects the Interpretation of RT-qPCR Results.
abstract: BACKGROUND
  Doxorubicin (DOX) acts in a variety of ways including DNA damage and enzyme inhibition, which consequently causes changes in gene expression of cells treated with this agent. Practical validation of experimental results followed by appropriate normalization of the factors investigated is crucial for obtaining biologically relevant results in gene expression studies.
  
  
  METHODS
  Six candidates were evaluated regarding their validity as internal reference genes: RPS23, FLOT2, UBB, ABCF1, ACTB, HPRT1. Optimization for quantitative polymerase reaction (qPCR) included: sensitivity, specificity, amplification efficiency and linear dynamic range determination. The gene expression stability was evaluated by real-time quantitative polymerase reaction (RT-qPCR) in two human cervical cancer cell lines: HeLa and DOX-resistant KB-V1 Cells treated under various concentrations of DOX.
  
  
  RESULTS
  DOX treatment changed gene expression and led to re-optimization of the cDNA template amounts. ACTB, HPRT1, RPS23 and FLOT2 are proposed to be sufficient as internal reference genes.
  
  
  CONCLUSIONS
  DOX may alter the reverse transcription and amplification reactions of RT-qPCR, thus creating a risk of misinterpretation of gene expression results.
authors.affiliation: National Medicines Institute, Department of Biochemistry and Biopharmaceuticals, Warsaw, Poland e.drozd@nil.gov.pl.
authors.forename: Ewa
authors.initials: E
authors.lastname: Drozd
authors.affiliation: National Medicines Institute, Department of Biochemistry and Biopharmaceuticals, Warsaw, Poland.
authors.forename: Jolanta
authors.initials: J
authors.lastname: Krzysztoń-Russjan
authors.affiliation: National Medicines Institute, Department of Biochemistry and Biopharmaceuticals, Warsaw, Poland.
authors.forename: Beata
authors.initials: B
authors.lastname: Gruber
mesh_terms: D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D017931:DNA Primers; D004317:Doxorubicin / Q000494:pharmacology*; D006367:HeLa Cells; D006801:Humans; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D048348:Reverse Transcription / Q000187:drug effects*; D013698:Templates, Genetic
chemical_list: D000903:Antibiotics, Antineoplastic; D017931:DNA Primers; D004317:Doxorubicin
keywords: Doxorubicin; HeLa; KB-V1; RT-qPCR; normalization