Housekeeping genes determination in a rat model of neurodegeneration induced by chronic expression of Tumor Necrosis Factor alpha (TNF-alpha)

JUAN CRUZ CASABONA; ANA DE LELLA EZCURRA; ARIEL CHERNOMORETZ; FERNANDO JUAN PITOSSI

Cordoba

Congreso; A2B2C Congress; 2011

A2B2C

Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) is a standard method to evaluate gene expression. In order to standardize the amount of starting biological material in these experiments (that can vary due to sample-to-sample variation, variation in RNA integrity, RT efficiency differences and cDNA sample loading variation), an elementary prerequisite is the determination of an internal control gene with assumed stable expression under any experimental condition. These genes are constitutively expressed and usually maintain cellular structure or homeostasis and are referred as housekeeping genes (HKG). To normalize the absolute quantification according to a single reference gene, a second set of qRT-PCR reactions has to be performed for the invariant HKG on all experimental samples. The relative abundance values are calculated for the internal control as well as for the target gene. For each target gene sample, the relative abundance value obtained is divided by the value derived from the control sequence in the corresponding target gene. The normalized values for different samples can then directly be compared [1]. Although unstable or inconsistent housekeeping gene expression will misrepresent experimental effects on target gene expression, housekeeping genes are often chosen arbitrarily rather than systematically. Recently an animal model of neurodegeneration in the substantia nigra (SN) was developed at our laboratory [2]. In the present model, neuronal demise is accomplished by chronic expression of soluble mouse TNF-alpha after intracranial injection in the SN of wistar rats of a recombinant adenovector (AdTNF-alpha) expressing this cytokine.The aim of the project in which this study is framed, is to identify mediator molecules induced by TNF-alpha that provide a unique effect in neuron viability, in this animal model. The experimental approach includes evaluation of overall gene expression by microarrays followed by technical validation of the candidate differential expressed genes. HKG identification is mandatory to accomplish this issue. In the present study, intracranial injection of AdTNF-alphawas performed, as well as intracranial injection of a control adenovirus, in two groups of young adult male wistar rats. 14 days after injection, SN samples were collected, homogenized and mRNA was extracted and transcribed into cDNA for each sample. HKG expression was evaluated for all samples using a set of 5 candidate genes by qRT-PCR. Efficiency corrected data of these assays were used to estimate candidate HKG expression stability using specific software (Bestkeeper, geNorm and NormFinder). In parallel global gene expression was evaluated by microarray assays, and the candidate HKG expression was compared to qRT-PCR data.