Article

Selection of internal reference genes for SYBR green qRT-PCR studies of rhesus monkey (Macaca mulatta) tissues.

Division of Biological Sciences, College of Natural Sciences, Pusan National University, Busan, 609-735, Republic of Korea.
BMC Molecular Biology (Impact Factor: 2.8). 10/2008; 9:78. DOI: 10.1186/1471-2199-9-78
Source: PubMed

ABSTRACT The rhesus monkey (Macaca mulatta) is a valuable and widely used model animal for biomedical research. However, quantitative analyses of rhesus gene expression profiles under diverse experimental conditions are limited by a shortage of suitable internal controls for the normalization of mRNA levels. In this study, we used a systematic approach for the selection of potential reference genes in the rhesus monkey and compared their suitability to that of the corresponding genes in humans.
Eight housekeeping genes (HKGs) (GAPDH, SDHA, ACTB, RPL13A, RPL32, UBA52, PGK1Y, and YWHAZ) from rhesus monkeys and humans were selected to test for normalization of expression levels in six different tissue types (brain, colon, kidney, liver, lung, and stomach). Their stability and suitability as reference genes were validated by geNorm, NormFinder and BestKeeper programs. Intriguingly, RPL13A and RPL32 were selected as ideal reference genes only in rhesus monkeys.
The results clearly indicated the necessity of using different reference genes for normalization of expression levels between rhesus monkeys and humans in various tissues.

0 Bookmarks
 · 
210 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) has been widely used to quantify relative gene expression because of the specificity, sensitivity, and accuracy of this technique. In order to obtain reliable gene expression data from RT-qPCR experiments, it is important to utilize optimal reference genes for the normalization of target gene expression under varied experimental conditions. Previously, we developed and validated a novel icv-STZ cynomolgus monkey model for Alzheimer's disease (AD) research. However, in order to enhance the reliability of this disease model, appropriate reference genes must be selected to allow meaningful analysis of the gene expression levels in the icv-STZ cynomolgus monkey brain. In this study, we assessed the expression stability of 9 candidate reference genes in 2 matched-pair brain samples (5 regions) of control cynomolgus monkeys and those who had received intracerebroventricular injection of streptozotocin (icv-STZ). Three well-known analytical programs geNorm, NormFinder, and BestKeeper were used to choose the suitable reference genes from the total sample group, control group, and icv-STZ group. Combination analysis of the 3 different programs clearly indicated that the ideal reference genes are and in the total sample group, and in the control group, and and in the icv-STZ group. Additionally, we validated the normalization accuracy of the most appropriate reference genes ( and ) by comparison with the least stable gene () using quantification of the and genes in the total sample group. To the best of our knowledge, this research is the first study to identify and validate the appropriate reference genes in cynomolgus monkey brains. These findings provide useful information for future studies involving the expression of target genes in the cynomolgus monkey.
    PLoS ONE 01/2013; 8(2):e56034. · 3.53 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The common marmoset (Callithrix jacchus) is increasingly being used as a non-human primate animal model in biomedical research. To perform accurate quantitative analysis of gene expression by quantitative reverse transcription polymerase chain reaction, reliable reference genes should be selected. In this study, we evaluated the expressions of 11 widely used reference genes: ACTB, ATP5F1, B2M, GAPDH, HPRT1, PGK1, PPIA, RN18S1, RPLP0, TBP and UBC in 12 tissues and five brain areas of healthy common marmosets. NormFinder and geNorm indicated that the most suitable reference genes for cross-sectional studies of the 17 tissues were RN18S1 and RPLP0. Conversely, ACTB and PPIA were the most suitable for analyzing brain samples; however, the expression of PGK1 fluctuated among brain areas. These results indicate that suitable reference genes differ between the tissues examined. This study provides fundamental information for gene expression studies of the common marmoset and highlights the importance of validating reference genes before quantification of target mRNAs.
    Molecular Biology Reports 09/2013; · 2.51 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Ozone, a pervasive environmental pollutant, adversely affects functional lung growth in children. Animal studies demonstrate that altered lung development is associated with modified signaling within the airway epithelial mesenchymal trophic unit, including mediators that can change nerve growth. We hypothesized that ozone exposure alters the normal pattern of serotonin, its transporter (5-HTT) and two key receptors (5-HT2A and 5-HT4); a pathway involved in postnatal airway neural, epithelial and immune processes. We exposed monkeys to acute or episodic ozone during the first 2 or 6 months of life. There were 3 exposure groups/age: 1) filtered air (FA), 2) acute ozone challenge (AO), and 3) episodic ozone+acute ozone challenge (EAO). Lungs were prepared for compartment-specific qRT-PCR, immunohistochemistry and stereology. Airway epithelial serotonin immunopositive staining increased in all exposure groups with the most prominent in 2 mo midlevel and 6 mo distal airways. Gene expression of 5-HTT, 5-HT2AR and 5-HT4R increased in an age dependent manner. Overall expression was greater in distal compared to midlevel airways. Ozone exposure disrupted both 5-HT2AR and 5-HT4R protein expression in airways and enhanced immunopositive staining for 5-HT2AR (2 mo) and 5-HT4R (6 mo) on smooth muscle. Ozone exposure increases serotonin in airway epithelium regardless of airway level, age and exposure history; and changes the spatial pattern of serotonin receptor protein (5-HT2A, 5-HT4) and 5-HTT gene expression depending on compartment, age and exposure history. Understanding how serotonin modulates components of reversible airway obstruction exacerbated by ozone exposure sets the foundation for developing clinically relevant therapies for airway disease.
    Toxicological Sciences 04/2013; · 4.33 Impact Factor

Full-text (2 Sources)

View
35 Downloads
Available from
Jun 5, 2014