[논문]Keeping house: evaluation of housekeeping genes for real-time PCR in the red alga, Bostrychia moritziana (Florideophyceae)

Shim, Junbo; Shim, Eunyoung; Kim, Gwang Hoon; Han, Jong Won; Zuccarello, Giuseppe C.

doi:10.4490/algae.2016.31.5.25

Keeping house: evaluation of housekeeping genes for real-time PCR in the red alga, Bostrychia moritziana (Florideophyceae) 원문보기

Algae, v.31 no.2, 2016년, pp.167 - 174

Shim, Junbo (Department of Biology, Kongju National University) , Shim, Eunyoung (Department of Biology, Kongju National University) , Kim, Gwang Hoon (Department of Biology, Kongju National University) , Han, Jong Won (National Marine Biodiversity Institute of Korea) , Zuccarello, Giuseppe C. (School of Biological Sciences, Victoria University of Wellington)

Abstract ▼ AI-Helper

Biological response of cells to variable conditions should affect the expression level of certain genes. Quantification of these changes in target genes needs stable internal controls. Real-time quantitative polymerase chain reaction (PCR) has traditionally used reference or ‘housekeeping’ genes, that are considered to maintain equal expression in different conditions, to evaluate changes in target genes between samples and experimental conditions. Recent studies showed that some housekeeping genes may vary considerably in certain biological samples. This has not been evaluated in red algae. In order to identify the optimal internal controls for real-time PCR, we studied the expression of eleven commonly used housekeeping genes; elongation factor 1-alpha, glyceraldehyde-3-phosphate dehydrogenase, β-actin, polyubiquitin, 30S ribosomal gene, 60S ribosomal gene, beta-tubulin, alpha-tubulin, translation initiation factor, ubiquitin-conjugating enzyme, and isocitrate dehydrogenase in different life-history stages of Bostrychia moritziana. Our results suggest that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 30S ribosomal gene, have the most stable gene expression levels between the different life history stages (male, female, carposporophyte, and tetrasporophyte), while the other genes are not satisfactory as internal controls. These results suggest that the combinations of GAPDH and 30S would be useful as internal controls to assess expression level changes in genes that may control different physiological processes in this organism or that may change in different life history stages. These results may also be useful in other red algal systems.

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제안 방법

2004). The expression stability of all candidate HKGs was evaluated by comparing the differences of observed Cq values across life-history stages. Pairwise variation (V_n/n+1) was calculated using geNorm.

성능/효과

moritziana in constant cul- ture conditions, the combination of GAPDH and 30S was the best gene set for this internal control. More than half of the tested HKGs including ACTB and 60S protein were no good for internal controls.
This could lead to erroneous conclusions on the changes in expression level in target genes. Our results showed that, at least be- tween life history stages of B. moritziana in constant cul- ture conditions, the combination of GAPDH and 30S was the best gene set for this internal control. More than half of the tested HKGs including ACTB and 60S protein were no good for internal controls.
crispus collected on the shore and stressed in situ. Their results showed that translation initiation factor 4A-2 and eukary- otic polypeptide chain release factor 3 were the best nor- malization genes and GAPDH was one of the worst genes for the internal control in stress-related experiment. Both C.
4). These results indicate that the combination of GAPDH and IcdH gives the best gene set for internal control in comparisons between the life history stages of B. moritziana.

후속연구

for each organism under study. Although RT-qPCR is expensive and time consuming a survey of potential candidate genes at the start of a study would be essential in reducing uncertainty, increasing confidence and may lessen the need for additional experiments to confirm any results.

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