Transcriptional expression study in the central nervous system of rats: what gene should be used as internal control?

Ana Carolina De Moura,Grasiela Agnes,Silvana Almeida,Márcia Giovenardi,Ana Beatriz Gorini Da Veiga,Virgínia Meneghini Lazzari

doi:10.1590/s1679-45082014ao3042

Ana Carolina De Moura, Grasiela Agnes + Show 4 more

Open Access

https://doi.org/10.1590/s1679-45082014ao3042

Copy DOI

Abstract

Objective A growing number of published articles report the expression of specific genes with different behavior patterns in rats. The levels of messenger ribonucleic acid transcripts are usually analyzed by reverse transcription followed by polymerase chain reaction and quantified after normalization with an internal control or reference gene (housekeeping gene). Nevertheless, housekeeping genes exhibit different expression in the central nervous system, depending on the physiological conditions and the area of the brain to be studied. The choice of a good internal control gene is essential for obtaining reliable results. This study evaluated the expression of three housekeeping genes (beta-actin, cyclophilin A, and ubiquitin C) in different areas of the central nervous system in rats (olfactory bulb, hippocampus, striatum, and prefrontal cortex).Methods Wistar rats (virgin females, n=6) during the diestrum period were used. Total ribonucleic acid was extracted from each region of the brain; the complementary deoxyribonucleic acid was synthesized by reverse transcription and amplified by real-time quantitative polymerase chain reaction using SYBR™ Green and primers specific for each one of the reference genes. The stability of the expression was determined using NormFinder.Results Beta-actin was the most stable gene in the hippocampus and striatum, while cyclophilin A and ubiquitin C showed greater stability in the prefrontal cortex and the olfactory bulb, respectively.Conclusion Based on our study, further studies of gene expression using rats as animal models should take into consideration these results when choosing a reliable internal control gene.

Highlights

IntroductionGene expression analysis requires precise and reproducible measurements of specific messenger ribonucleic acid (mRNA) sequences
Structures in the central nervous system (CNS) such as olfactory bulb (OB), hippocampus (HP), striatum (ST), prefrontal cortex (PFC), posterodorsal medial amygdala (MePD), and medial preoptic area (MPOA) are responsible for the appearance and maintenance of different behaviors.[1,2] Understanding the molecular mechanisms involved in the regulation of signaling pathways in the CNS has been the basis of many studies, which aimed to address how a pattern of behavior is controlled by the expression of a candidate gene or group of genes.[3,4,5] gene expression assays have been increasingly employed in behavioral studies using animal models, based on analysis of specific transcripts in the CNS and their association with different patterns of behavior
In some gene expression studies in the CNS, ActB was shown to be as stable as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), but its stability depends on the tissue and conditions used in the analyses.[21,22] Chen et al[23] found that other constitutive genes, such as eukaryotic translation elongation factor (EF) and GAPDH, are more stable than ActB in some areas of the brain such as the auditory cortex and the cochlea of rats, and should be considered as reference genes in quantitative gene expression analyses in these regions

Summary

Introduction

Gene expression analysis requires precise and reproducible measurements of specific messenger ribonucleic acid (mRNA) sequences. The most common method used to quantify mRNA is the amplification of individual RNA molecules by combining reverse transcription and real-tim e polymerase chain reaction (RT-PCR),(6) which enables a sensitive and accurate quantification of mRNA expression levels. Most gene expression experiments require ribonucleic acid (RNA) isolation and processing, and the final amount of RNA may vary among samples. Performing RT-PCR analysis requires controlled parameters to obtain reliable quantitative expression measures. These include variations in initial sample amount, RNA recovery, RNA integrity, efficiency of complementary deoxyribonucleic acid (cDNA) synthesis, and differences in the overall transcriptional activity of the tissues or cells analyzed.[7,8]

Methods

Discussion

Conclusion