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?ConclusionsCaution 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.

SUBMITTER: Sharungbam GD 

PROVIDER: S-EPMC3422209 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature