Potential utility of 14 candidate housekeeping genes as normalization reference for RT-qPCR analysis with developmental samples (fertilized eggs to late veliger larvae) in Pacific abalone Haliotis discus hannai was evaluated using four different statistical algorithms (geNorm, NormFinder, BestKeeper and comparative ΔCT method). Different algorithms identified different genes as the best candidates, and geometric mean-based final ranking from the most to the least stable expression was as follow: RPL5, RPL4, RPS18, RPL8, RPL7, UBE2, RPL7A, GAPDH, RPL36, PPIB, EF1A, ACTB and B-TU. The findings were further validated via relative quantification of metallothionein (MT) transcripts using the stable and unstable reference genes, and expression levels of MT were greatly influenced according to the choice of reference genes. In overall, our data suggest that RPL5 and RPS18, either singly or in combination, are appropriate for normalizing gene expression in developmental samples of this abalone species, whereas ACTB, B-TU and EF1A are less stable and not recommended. In addition, our findings propose that standard deviations in geometric ranking as well as geometric mean itself should also be taken into account for the final selection of reference gene(s). This study could be a useful basis to facilitate the generation of accurate and reliable RT-qPCR data with developmental samples in this abalone species.

Honey bee, Apis mellifera L., have been widely used as a model organism for biological science because of its highly developed sociality, specialized labor division and passive population management. In order to examine the expression patterns of genes putatively involved in social development in honey bee, quantitative real-time PCR (qRT-PCR) that has been widely used to investigate the expression level of target gene can be used in honey bee study. However, the selection and validation of optimal reference genes is a crucial step prior to running qRT-PCR. In the present study, therefore, the seasonal expression stability of five candidate reference genes in the abdomen of forager and nurse was investigated using three programs (geNorm, NormFinder and BestKeeper), and selected reference genes were validated by the normalization of expression level of vg encoding vitellogenin. Although three programs revealed slightly different gene stability values, overall the combination of two genes (rpS18 and gapdh encoding ribosomal protein S18 and glyceraldehyde-3-phosphate dehydrogenase, respectively) was resulted in the most suitable use for normalization of the target gene in forager. However, a single gene, either rpL32 or rpS18 in the nurse or either rpL32, rpS18, or gapdh in the comparison between foragers and nurses, were suggested to be applied for normalization of seasonal and labor-specific gene expression by qRT-PCR.

The fruit fly, Drosophila melanogaster, is a good model organism in various areas of biological science. Since D. melanogaster has been thought to be adapted to the chemical stress environment caused by the overripen, decay and fermented fruits, identification of the genes involved in chemical tolerance and investigation of their expression patterns are essential for better understanding of the physiological evolution in D. melanogaster. For investigation of the gene expression level, quantitative real-time PCR (qRT-PCR) can be applied to quantify gene expression level and selection of reliable reference gene(s) for normalization is an accurate step. In the present study, therefore, we validated the expression stabilities of ten candidate reference genes using three softwares (geNorm, NormFinder and BestKeeper) in D. melanogaster exposed to different concentrations of acetic acid, ethanol and 2-phenylethanol. Although three programs resulted in slightly different gene stability ranks, but overall tbp encoding TATA box binding protein was most stable gene in acetic acid and ethanol exposed fly, while nd encoding NADH dehydrogenase was the most suitable reference gene in the case of 2-phenylethanol treatment. In the comparison of three chemical treatment condition, nd was also suggested to be most optimal reference gene. In addition, optimal number of reference gene for accurate normalization was calculated by geNorm pairwise analysis, and selection of multiple reference genes was suggested to be better for target gene normalization method than use of a single reference gene.

과일이나 농작물의 부패 및 발효 환경에서는 Methanol, Ethanol, Acetic acid을 비롯한 다양한 화학물질들이 생산된다. Drosophila melanogaster는 이러한 발효·부패 환경에 서식하면서 일정 농도 이상의 다양한 화학물질에 지속적으로 노출되어 생존하도록 적응되어온 것으로 생각된다. 다양한 화학물질이 포함한 환경에 안정적으로 서식하기 위해서는 D. melanogaster는 화학물질에 능동적으로 반응하여 해독 유전자나 대사 관련 유전자의 발현량을 변화 시킴으로써 발효·부패 환경에서 생성되는 화학물질에 대한 높은 내성을 가지고 있을 것으로 판단된다. 현재까지 유전자의 발현량 측정을 위해 real-time PCR를 이용하여 reference gene의 발현량을 기준으로 정량화하는 방법이 가장 널리 사용되고 있다. 그러나 조직별, 환경별, 발달단계를 비롯한 다양한 조건에서 안정적으로 발현되는 reference 유전자 선정이 필수적으로 선행되어야 하므로 본 연구에서는 발효·부패 환경에서 생산되는 두 화학물질인 Methanol과 Ethyl Acetate에 노출된 D. melanogaster에서 안정적으로 발현되는 reference gene을 찾는 연구를 실시하였다. 본 연구에서는 다양한 농도의 Methanol과 Ethyl Acetate을 D. melanogaster에 노출시킨 후 RNA 추출과 cDNA 합성을 실시였고, 5가지 후보 reference gene (hsp22, nd, rpL18, tbp and ef-1b)의 안정적 발현 여부를 qRT-PCR을 통해 조사하였으며, 유전자 발현의 안정성을 측정하는 3가지 프로그램(geNorm, NormFinder, BestKeeper)을 이용해 비교·분석하였다. 본 학회에서는 연구의 과정과 그 결과를 발표하고자 한다.

Honey bee has been widely used as a model insect for biological sciences because of its sociality and specialized labor division. For the investigation of the seasonal and labor-dependent expression patterns of genes putatively involved in its sociality, quantitative real-time PCR (qRT-PCR) can be applied to quantify gene expression level and selection of reliable reference gene(s) for normalization is an accurate step. In this study, using three softwares (geNorm, NormFinder and BestKeeper), we evaluated seasonal expression stabilities of four reference genes that have been widely used for qRT-PCR in forager and nurse heads. Among four candidates, two genes, rpS18 and gapdh, were suggested to be the optimal reference genes for qRT-PCR.

Watermelon (Citrullus lanatus) is one of the most economically important cucurbitaceous crop over the world. Screening of proper reference genes was needed to reverse transcription quantitative real-time PCR (qRT-PCR), and it is bausic step of many researches including gene expression analysis. However, the reference genes on watermelon has not yet been reported systematically. Therefore, eight candidates of reference genes were selected with reference to Arabidopsis or cucumber papers. They are β-Actin, elongation factor 1-α, glyceraldehy-3-phosphate-dehydrogenase, NADP-isocitrate dehydrogenase, leunig, polypyrimidine tract-binding protein1, ubiquitin-conjugating eznyme E2, and 18S ribosomal RNA. The expression levels of genes were evaluated by qRT-PCR under biotic stress (Colletotrichum orbiculare treatment), plant hormone treatment (100 μM ABA), and abiotic stresses such as drought, cold (4℃), salt (250 mM NaCl) stresses. We founded appropriate reference genes which did not induce or reduce gene expression levels under broad spectrum of stresses by qRT-PCR analysis. These results may provide proper information for the use of appropriate reference genes for gene expression studies in watermelon qRT-PCR analysis.