선택한 단어 수는 입니다.
최소 단어 이상 선택하여야 합니다.
최소 단어 이상 선택하여야 합니다.
선택한 단어 수는 30입니다.
최대 10 단어까지만 선택 가능합니다.
최대 10 단어까지만 선택 가능합니다.
문제 정의
- However, despite its importance, the evaluation of stable reference genes from this abalone species in relation with heavy metal exposures has been little studied. Based on this need, the objective of this study was to validate different HKGs associated with heavy metal exposures as the initial step in environmental toxicogenomics of this abalone species.
제안 방법
- LightCycler 480 Real-Time PCR System (Roche Life Science, Mannheim, Germany) was used for all the qPCR experiments in this study. Based on a series of preliminary evaluations, the optimal pair of primers was selected for each gene for further use in expression stability analysis. The summarized information on the genes and oligonucleotide primers used in the present study is provided in Table 1.
- Based on the evaluation with four different statistical approaches (geNorm, NormFinder, BestKeeper, and ΔCT method) followed by ranking decision with the RefFinder tool, all the four approaches did not recommend the same reference gene for the most suitable internal control gene (Table 2).
- For NormFinder, the same input file format as the geNorm was adopted; however, Cq values were converted into relative quantities after correcting the PCR efficiencies. For BestKeeper, untransformed Ct values and PCR efficiency (E) were used to determine the best suited standards and to combine them into an index by the coefficient of determination and the P value based on the geometric mean of the Cq values. The ΔCT method compares the relative expression of pairs of genes within each sample to confidently identify useful HKGs.
- In order to evaluate the expression stability of candidate reference genes in either tissue type, the first dataset comprised of gill samples (n = 84 including biological replications), while the second dataset consisted of 84 hepatopancreas samples from non-exposed and metal-exposed groups. For each biological sample (i.e., individual cDNA sample) from either tissue types, technical replications of the qPCR assay were made in triplicates to determine the median value of each biological sample, and afterward, the control cycle threshold (Ct) values (i.e., biological replications) within an exposure treatment set were averaged in order to prevent the proportion of data from non-exposed control groups from being too high in the raw datasets.
- For each gene, multiple primer pairs were designed considering primer length (20–22 bp), G+C contents (40–60 %), melting temperature (55–65 ℃), and amplicon size (100–200 bp).
- 2011), and the hepatopancreas is the main organ responsible for deposits and metabolic detoxification of trace metals in aquatic invertebrates (Rainbow 2007). In this study, we performed two sets of heavy metal exposures (i.e., exposure to different heavy metals, Cu, Zn, Cd, and exposure to different doses of a given metal, Cu), and the raw RTqPCR data were pooled into two subsets of tissue types (gill and hepatopancreas) in order to evaluate the expression stability of candidate reference genes in a tissue-specific fashion. The exposure conditions similar with those in the present study have already been proven to induce pro-oxidant stresses in abalone species belonging to Haliotidae (Kim et al.
- 2006), based on the standard curve prepared with 4-log serial dilution points of abalone complementary DNA (cDNA) samples. LightCycler 480 Real-Time PCR System (Roche Life Science, Mannheim, Germany) was used for all the qPCR experiments in this study. Based on a series of preliminary evaluations, the optimal pair of primers was selected for each gene for further use in expression stability analysis.
- 2010). Twelve candidates selected in the present study are assigned to several categories of functional ontology such as cytoskeletal cell shape and mobility (ACTB and B-TU), energy metabolism (GAPDH), translation and protein synthesis (EF1A and various RPLs), and protein metabolism and degradation (UBE2). Such a selection regime might also be of importance for adopting software programs and interpreting outputs, since the algorithms used by each program differ from one another.
- With the geNorm software program, the minimum number of reference genes for accurate normalization was determined based on the calculation of the pairwise variation Vn/Vn + 1 between two sequential normalization factors to consider the necessity of adding the next reference gene, in which large variation means that the newly added gene should have a significant effect on the normalization accuracy and hence be added for calculation. Considering the recommended cutoff value of 0.
- SYL carried out the biological stimulatory experiments using abalones and all the gene expression studies. YKN designed the study, performed bioinformatics analysis, and drafted the manuscript. All authors read and approved the final manuscript.
대상 데이터
- Based on searches against both a local transcriptome next-generation sequencing (NGS) database constructed with abalone tissues (unpublished data) and the public NCBI GenBank database (http://www.ncbi.nlm.nih.gov), 12 candidate HKGs were selected. They included cytoskeletal β-actin (ACTB), β-tubulin (B-TU), elongation factor-1 alpha (EF1A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), peptidyl-prolyl cis-trans isomerase B (PPIB), ubiquitin-conjugating enzyme E2 (UBE2), and six kinds of ribosomal proteins (RPL3, RPL4, RPL5, RPL7, RPL7A, and RPL8).
- Fig. 1 Representative end-point gel showing the specific amplification of the single PCR band for each of 12 candidate reference genes tested in this study. PCR products were run on 2 % agarose gel and visualized by ethidium-bromide staining.
- 5 cm) were exposed to cadmium (Cd), copper (Cu), or zinc (Zn). Heavy metal chemicals (analytical grade) were used (Sigma-Aldrich, St. Louis, MO, USA). Six individuals were assigned to one of three 50-L tanks containing 40 L seawater supplemented with each heavy metal.
- In the present study, we selected gill and hepatopancreas tissues, the two main organs with respect to the uptake and bioprocess of heavy metal ions in aquatic organisms. In order to evaluate the expression stability of candidate reference genes in either tissue type, the first dataset comprised of gill samples (n = 84 including biological replications), while the second dataset consisted of 84 hepatopancreas samples from non-exposed and metal-exposed groups. For each biological sample (i.
- Louis, MO, USA). Six individuals were assigned to one of three 50-L tanks containing 40 L seawater supplemented with each heavy metal. The dose strength of each heavy metal (Cd, Cu, or Zn) was 0.
- 25 ppm) for different durations of 24, 48, and 72 h. Twelve abalones were assigned into either one of four tanks (two replicate tanks for 0 and 0.25 ppm). Again, all other tank conditions were identically prepared with those mentioned above.
데이터처리
- Calculated levels of MT transcripts in gill and hepatopancreas based on each normalization regime were subjected to one-way ANOVA followed by Duncan’s multiple range tests in order to examine statistical significance at P = 0.05.
- Finally, comprehensive ranking to identify the most appropriate control gene was determined using the software RefFinder (http://fulxie.0fees.us/?type=reference&ckattempt=1), in which the four abovementioned software programs were integrated to compare the ranking of the tested candidate reference genes.
성능/효과
- NormFinder also recommended RPL7, followed by RPL8, for the most stable references while indicated BTU as the most variable gene. According to BestKeeper, RPL7 and RPL8 turned out to be the most stable in the gill, whereas B-TU and UBE2 were pointed as the least stable candidates, although all the 12 genes tested showed SD values lower than 1. For the most stable candidate genes, according to the ΔCT method, the RPL7 gene was the first gene of the rank in the gill, while BTU was the last gene.
- , upregulation or downregulation) was not affected by the kinds of reference genes tested. Collectively, the overall findings from this study confirm not only that the reference genes chosen under a certain experimental treatment conditions may not be universally applicable to other conditions but also that expression stability of a given reference gene could not have consensus across tissue types.
- Of the 12 candidate reference genes tested, B-TU was the least expressed (the highest mean Cq values) gene in the gill subsets, whereas RPL7 was the most expressed gene in this tissue type. Considering the variation among samples in a given gill tissue, B-TU, EF1A, GAPDH, RPL3, RPL5, and UBE2 exhibited relatively greater variation in their expression levels than other candidate genes. In the subset of the hepatopancreas also, B-TU and RPL7 showed the least and highest expression levels, respectively, as similarly in the gill, although their absolute mean Cq values were different from those observed in the subset of the gill (Fig.
- Also, every primer pair used in the present qPCR assays successfully revealed a single peak from the melting curve analysis (data not shown), indicating that the specific amplification for each candidate gene segment could be achieved. Of the 12 candidate reference genes tested, B-TU was the least expressed (the highest mean Cq values) gene in the gill subsets, whereas RPL7 was the most expressed gene in this tissue type. Considering the variation among samples in a given gill tissue, B-TU, EF1A, GAPDH, RPL3, RPL5, and UBE2 exhibited relatively greater variation in their expression levels than other candidate genes.
- As shown in Table 2, the recommended comprehensive ranking of the stability in the gill was determined as RPL7 > RPL8 > ACTB > RPL3 > PPIB > RPL7A > EF1A > RPL4 > GAPDH > RPL5 > UBE2 > B-TU. Overall, the results indicate RPL7 could be considered as the ideal reference gene in the gill regarding metal exposure experiment in this abalone species, while UBE2 and B-TU would be the worst or unstable references that are not recommended for the use in this case.
- , ACTB and GAPDH) but excluded the 18S rRNA, one of the most popular reference genes. The reason of exclusion was that 18S rRNA is not a poly(A+)-tailed RNA; therefore, the random priming for RT reaction is, at least in part, mandatory and the oligo-d(T) priming may not be applicable to this RNA type. In this case, due to its extremely high abundance, signal saturation obtained at very low Ct values might often be problematic unless template cDNA for the samples analyzed with 18S primers were diluted at least a hundredfold relative to all other candidate genes.
후속연구
- Our results also highlight the importance of selecting the suitable reference gene(s) for RT-qPCR studies, considering not only the experimental conditions but also tissue types under evaluation. Data from this study could be a good fundamental basis to guide future design of RT-qPCR studies with respect to metal regulation/detoxification and other related physiological aspects in this abalone species.
※ AI-Helper는 부적절한 답변을 할 수 있습니다.