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Comparative Study
. 2008 Sep 10:9:78.
doi: 10.1186/1471-2199-9-78.

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

Kung Ahn  1 , Jae-Won Huh, Sang-Je Park, Dae-Soo Kim, Hong-Seok Ha, Yun-Ji Kim, Ja-Rang Lee, Kyu-Tae Chang, Heui-Soo Kim
Affiliations
Comparative Study

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

Kung Ahn et al. BMC Mol Biol. .

Abstract

Background: 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.

Results: 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.

Conclusion: 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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Figures

Figure 1
Figure 1
Selection of the most suitable reference genes for normalization in rhesus monkey (a and b) and human (c and d) samples using geNorm analysis. Stepwise exclusion of the least stable genes by calculating the average expression stability (a and c) was conducted. The value of M was calculated for each gene, and the least stable gene with the highest M value was automatically excluded for the next round of calculations. The x-axis from left to right indicates the ranking of the genes according to their expression stability. Determination of the optimal number of reference genes for normalization (b and d) was conducted. The software calculates the normalization factor from at least two genes at which the variable V defines the pair-wise variation between two sequential normalization factors.
Figure 2
Figure 2
Logarithmic histogram of the expression levels of 8 internal reference genes determined in 6 different tissues. Relative expression levels of rhesus monkey (A) and human (B) with six different tissues. The respective bar and genes name are: actin beta (ACTB: light blue bar), Glyceraldehyde-3-phospate dehydrogenase (GAPDH: light purple bar), Hydroxymethylbilane synthase (HMBS: light green bar), Hypoxanthine phosphoribosyltransferas 1 (HPRT1: green bar) Ribosomal protein L13A (RPL13A: dark purple bar), 60S ribosomal protein L32 (RPL32: pink bar), TATA box binding protein (TBP: dark blue bar), Tyrosine 3-monooxygenase (YWHAZ: sky blue bar).
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