Selection of internal reference genes for SYBR green qRT-PCR studies of rhesus monkey (

Division of Biological Sciences, College of Natural Sciences, Pusan National University, Busan, 609-735, Republic of Korea.
BMC Molecular Biology (Impact Factor: 2.19). 10/2008; 9(1):78. DOI: 10.1186/1471-2199-9-78
Source: PubMed


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.

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Available from: Sang-Je Park, Oct 09, 2015
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    • "Transcripts for the ribosomal proteins, RPL32 and RPL13A, have been identified recently as reference genes for use in real-time PCR analyses performed on macaque tissues (Ahn et al., 2008). To identify and validate a suitable housekeeping gene for the macaque endometrium, simplex reactions were performed to determine the constitutive expression profiles of 18S rRNA, 28S rRNA, RPL32 mRNA, MRSP10 mRNA and PPIA mRNA, with methods similar to those described by Nolan et al. (2006). "
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    ABSTRACT: Endothelins (EDNs) are thought to modulate endometrial blood flow during menses, stromal healing and endometrial growth during the proliferative phase. Our goal was to assess the effects of estrogen and progesterone on the EDN paracrine system in the endometrium of rhesus macaques. In this study, archived samples were used. These samples were collected from oophorectomized rhesus macaques that were treated sequentially with estradiol (E(2)) and then E(2) plus progesterone to create artificial menstrual cycles. Endometrium from animals in the menstrual, proliferative and secretory phases of the artificial cycle were analyzed by real-time PCR, in situ hybridization and immunocytochemistry to detect changes in EDN peptides (EDN1, EDN2, EDN3), EDN receptors (EDNRA, EDNRB), EDN-converting enzyme 1 (ECE1) and membrane metalloendopeptidase (MME)-an enzyme that degrades the EDNs. Compared with the late secretory phase, progesterone withdrawal at the end of the artificial menstrual cycle triggered an increase (P< 0.05) in EDN1, EDNRB and ECE1 in the upper functionalis zone during menses of the next cycle. Treatment with E(2) alone in the proliferative phase increased (P< 0.05) EDNRA transcript, which was confined predominantly to the stromal cells. E(2) plus progesterone in the artificial secretory phase suppressed (P< 0.05) the expression of EDN3 in the functionalis zone stroma and epithelia, tended (P= 0.08) to attenuate levels of epithelial EDN2 and markedly up-regulated (P< 0.05) the stromal expression of MME. Our results indicate that estrogen and progesterone regulate the EDN family during the menstrual cycle. The changes in the EDN paracrine system during the mid-secretory phase may indicate a role for EDN during embryo implantation.
    Human Reproduction 07/2011; 26(7):1715-28. DOI:10.1093/humrep/der115 · 4.57 Impact Factor
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    • "The SYBR Green amplifications were performed as followed: initial denaturation at 95°C for 20 s followed by 40 cycles of 3 s at 95°C and 30 s at 60°C. Q-RT-PCR data were normalized to the stably expressed HPRT mRNA for NHP samples [36], [37]. For murine samples, 11 potential reference genes were evaluated using the Mouse Endogenous Control Gene Panel kit (Tataa biocenter). "
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    ABSTRACT: Recombinant adeno-associated viral (rAAV) vectors can support long-term transgene expression in quiescent tissues. Intramuscular (i.m.) administration of a single-stranded AAV vector (ssAAV) in the nonhuman primate (NHP) results in a peak protein level at 2-3 months, followed by a decrease over several months before reaching a steady-state. To investigate transgene expression and vector genome persistence, we previously demonstrated that rAAV vector genomes associate with histones and form a chromatin structure in NHP skeletal muscle more than one year after injection. In the mammalian nucleus, chromatin remodeling via epigenetic modifications plays key role in transcriptional regulation. Among those, CpG hyper-methylation of promoters is a known hallmark of gene silencing. To assess the involvement of DNA methylation on the transgene expression, we injected NHP via the i.m. or the intravenous (i.v.) route with a recombinant ssAAV2/1 vector. The expression cassette contains the transgene under the transcriptional control of the constitutive Rous Sarcoma Virus promoter (RSVp). Total DNA isolated from NHP muscle and liver biopsies from 1 to 37 months post-injection was treated with sodium bisulfite and subsequently analyzed by pyrosequencing. No significant CpG methylation of the RSVp was found in rAAV virions or in vector DNA isolated from NHP transduced tissues. Direct de novo DNA methylation appears not to be involved in repressing transgene expression in NHP after gene transfer mediated by ssAAV vectors. The study presented here examines host/vector interactions and the impact on transgene expression in a clinically relevant model.
    PLoS ONE 06/2011; 6(6):e20881. DOI:10.1371/journal.pone.0020881 · 3.23 Impact Factor
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    • "Thus, selection process of an ideal reference gene is critical for applicability of PCR in research. However, commonly used reference genes for tissue and cell-based PCR normalization such as glyceraldehydes-3-phosphate dehydrogenase (GAPDH), β-actin (Actb) and 18S ribosomal RNA (18S rRNA) are frequently applied without appropriate validation for their gene expression stability [4-6]. "
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    ABSTRACT: The accuracy of quantitative real-time RT-PCR (qRT-PCR) is often influenced by experimental artifacts, resulting in erroneous expression profiles of target genes. The practice of employing normalization using a reference gene significantly improves reliability and its applicability to molecular biology. However, selection of an ideal reference gene(s) is of critical importance to discern meaningful results. The aim of this study was to evaluate the stability of seven potential reference genes (Actb, GAPDH, 18S rRNA, CycA, Hprt1, Ywhaz, and Pgk1) and identify most stable gene(s) for application in tissue culture research using the rat and rabbit intervertebral disc (IVD). In vitro, four genes (Hprt1, CycA, GAPDH, and 18S rRNA) in rat IVD tissue and five genes (CycA, Hprt1, Actb, Pgk1, and Ywhaz) in rabbit IVD tissue were determined as most stable for up to 14 days in culture. Pair-wise variation analysis indicated that combination of Hprt1 and CycA in rat and the combination of Hprt1, CycA, and Actb in rabbit may most stable reference gene candidates for IVD tissue culture. Our results indicate that Hprt1 and CycA are the most stable reference gene candidates for rat and rabbit IVD culture studies. In rabbit IVD, Actb could be an additional gene employed in conjunction with Hprt1 and CycA. Selection of optimal reference gene candidate(s) should be a pertinent exercise before employment of PCR outcome measures for biomedical research.
    BMC Research Notes 05/2011; 4(1):162. DOI:10.1186/1756-0500-4-162
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