title: Identification of stable endogenous control genes for transcriptional profiling of photon, proton and carbon-ion irradiated cells.
abstract: BACKGROUND
  Quantitative analysis of transcriptional regulation of genes is a prerequisite for a better understanding of the molecular mechanisms of action of different radiation qualities such as photon, proton or carbon ion irradiation. Microarrays and real-time quantitative RT-PCR (qRT-PCR) are considered the two cornerstones of gene expression analysis. In interpreting these results it is critical to normalize the expression levels of the target genes by that of appropriately selected endogenous control genes (ECGs) or housekeeping genes. We sought to systematically investigate common ECG candidates for their stability after different radiation modalities in different human cell lines by qRT-PCR. We aimed to identify the most robust set of ECGs or housekeeping genes for transcriptional analysis in irradiation studies.
  
  
  METHODS
  We tested the expression stability of 32 ECGs in three human cancer cell lines. The epidermoid carcinoma cells (A431), the non small cell lung carcinoma cells (A549) and the pancreatic adenocarincoma cells (BxPC3) were irradiated with photon, proton and carbon ions. Expression Heat maps, clustering and statistic algorithms were employed using SUMO software package. The expression stability was evaluated by computing: mean, standard deviation, ANOVA, coefficient of variation and the stability measure (M) given by the geNorm algorithm.
  
  
  RESULTS
  Expression analysis revealed significant cell type specific regulation of 18 out of 32 ECGs (p < 0.05). A549 and A431 cells shared a similar pattern of ECG expression as the function of different radiation qualities as compared to BxPC3. Of note, the ribosomal protein 18S, one of the most frequently used ECG, was differentially regulated as the function of different radiation qualities (p ≤ 0.01). A comprehensive search for the most stable ECGs using the geNorm algorithm identified 3 ECGs for A431 and BxPC3 to be sufficient for normalization. In contrast, 6 ECGs were required to properly normalize expression data in the more variable A549 cells. Considering both variables tested, i.e. cell type and radiation qualities, 5 genes-- RPLP0, UBC, PPIA, TBP and PSMC4-- were identified as the consensus set of stable ECGs.
  
  
  CONCLUSIONS
  Caution is warranted when selecting the internal control gene for the qRT-PCR gene expression studies. Here, we provide a template of stable ECGs for investigation of radiation induced gene expression.
authors.affiliation: Molecular RadioOncology [E210], National Center for Tumor Disease (NCT), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 460, 69120 Heidelberg, Germany.
authors.forename: Geeta D
authors.initials: GD
authors.lastname: Sharungbam
authors.forename: Christian
authors.initials: C
authors.lastname: Schwager
authors.forename: Sara
authors.initials: S
authors.lastname: Chiblak
authors.forename: Stephan
authors.initials: S
authors.lastname: Brons
authors.forename: Lynn
authors.initials: L
authors.lastname: Hlatky
authors.forename: Thomas
authors.initials: T
authors.lastname: Haberer
authors.forename: Jürgen
authors.initials: J
authors.lastname: Debus
authors.forename: Amir
authors.initials: A
authors.lastname: Abdollahi
mesh_terms: D045744:Cell Line, Tumor; D015870:Gene Expression / Q000528:radiation effects*; D020869:Gene Expression Profiling / Q000379:methods*; D020043:Genes, Essential / Q000528:radiation effects*; D063193:Heavy Ion Radiotherapy; D006801:Humans; D017785:Photons; D011522:Protons; D011878:Radiotherapy / Q000009:adverse effects / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D011522:Protons