RESUMO
Coturniculture has been standing out as an industrial poultry activity in several countries around the world because of the several adaptive advantages of quails. Research that considers the analysis of gene expression can enhance this activity. This study aimed to analyze the stability of reference genes (RGs) in different tissues of quails (both males and females) for the recommendation of use in gene expression studies by the quantitative reverse transcription-polymerase chain reaction (RT-qPCR). The expression stability of ten RGs (ACTA1, ACTB, B2M, GAPDH, HMBS, SDHA, HPRT1, MRPS27, MRPS30, and RPL5) was analyzed in four tissues (breast muscle, abdominal fat, liver, and intestine), and assessed using the statistical tools geNorm, NormFinder, comparative ΔCq method, and BestKeeper. The HPRT1 gene was the most stable in all quail tissues tested, followed by MRPS27 and MRPS30 in breast muscle, B2M and RPL5 in abdominal fat, HMBS and B2M in the liver, and RPL5 and HMBS in the intestine. These results may help studies using RT-qPCR assays to assess quail tissues from both sexes because they provide data on the most stable genes, which should be tested as candidate RGs for other experimental conditions.
Assuntos
Perfilação da Expressão Gênica/normas , Codorniz/genética , Reação em Cadeia da Polimerase em Tempo Real/normas , Padrões de Referência , Animais , Mama/metabolismo , Feminino , Mucosa Intestinal/metabolismo , Fígado/metabolismo , Masculino , Reação em Cadeia da Polimerase em Tempo Real/métodosRESUMO
Understanding into acerola (Malpighia emarginata) molecular and biochemical bases is still obscure, despite it is one of the most important natural source of vitamin C for humans. Recently, our research group published the first data on acerola transcriptome generating valuable information to identify reference genes for RT-qPCR in this species. Hence, this study aimed to identify the most stably expressed genes based on acerola transcriptome data, and further to evaluate the suitability of F-box, U3, Merad50-ATPase, TGD4, NOB1, PA-RNA, RCC1, RBL and PGAL candidates for accurate gene expression normalization in leaf, flower and fruit at 12, 16 and 20 days after anthesis using RT-qPCR analysis. Three algorithms, geNorm, NormFinder, and BestKeeper confirmed the expression stability of all nine candidate reference genes, whereas RefFinder consensually summarized a comprehensive gene ranking. Based on geNorm, the combination of the most stable reference genes RBL and U3 for leaf/flower group, TGD4, F-box and PGAL (fruit developmental stages or fruit/leaf), RCC1, PGAL and RBL (fruit/flower) and RCC1, RBL, TGD4 and PGAL (total samples) were required for accurate normalization. Moreover, the use of these reference genes to assess the expression profile of GMP1 and NAT3 genes confirmed the reliability of ranking and defined the best combination of genes recommended by geNorm and RefFinder. This work will benefit further RT-qPCR studies in these acerola organs by offering a foundation for accurate normalization of gene expression profiling.
Assuntos
Perfilação da Expressão Gênica/normas , Malpighiaceae/genética , Transcriptoma/genética , Algoritmos , Flores/genética , Frutas/genética , Expressão Gênica/genética , Regulação da Expressão Gênica de Plantas/genética , Genes de Plantas/genética , Folhas de Planta/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
Roundup is a glyphosate-based herbicide (GBH) widely used in agriculture and may cause toxic effects in non-target organisms. Model organisms, as zebrafish, and analysis of gene expression by reverse transcription-quantitative PCR (RT-qPCR) could be used to better understand the Roundup toxicity. A prerequisite for RT-qPCR is the availability of appropriate reference genes; however, they have not been described for Roundup-exposed fish. The aim of this study was to evaluate the expression stability of six reference genes (rpl8, ß-act, gapdh, b2m, ef1α, hprt1) and one expressed repetitive element (hatn10) in organs of males (brain, gill, testis) and females (ovary) of zebrafish exposed to Roundup WG at three concentrations (0.065, 0.65 and 6.5 mg N-(phosphonomethyl) glycine/l) for 7 days. Genes were ranked by geNorm, NormFinder, BestKeeper, Delta C t and RefFinder, and their best combinations were determined by geNorm and NormFinder programs. The two most stable ranked genes were specific to each organ: gill (ß-act; rpl8); brain (rpl8; ß-act); testis (ef1α; gapdh); and ovary (rpl8; hprt1). The cat transcript level was used to evaluate the effect of normalization with these reference genes. These are the first suitable reference genes described for the analysis of gene expression in organs of Roundup-exposed zebrafish, and will allow investigations of the molecular mechanisms of Roundup toxicity.
Assuntos
Perfilação da Expressão Gênica , Glicina/análogos & derivados , Reação em Cadeia da Polimerase em Tempo Real , Peixe-Zebra , Animais , Feminino , Glicina/toxicidade , Herbicidas , Masculino , Padrões de Referência , Transcrição Reversa , Peixe-Zebra/genética , GlifosatoRESUMO
Golden pompano (Trachinotus ovatus) is an important economically fish species. In this study, with an aim to identify reliable reference genes for quantitative real-time PCR (qRT-PCR) in golden pompano, we evaluated the expression stability of eight housekeeping genes in the presence and absence of poly I:C stimulation in eight tissues. The PCR data was analyzed by geNorm and NormFinder algorithms. The results showed that the expression of all the examined genes exhibited tissue-dependent variations. When under normal physiological condition, geNorm and NormFinder identified B2M and 18S as suitable genes. When studying gene expression under conditions of poly I:C stimulation, the selection of the internal controls should be selected on a tissue basis. At 12 h stimulation, geNorm ranked Actin/UBCE, Actin/B2M, UBCE/B2M, Actin/UBCE, RPL13/B2M, UBCE/GAPDH, B2M/RPL13, and UBCE/B2M, respectively, as the most stably expressed genes in liver, spleen, kidney, gill, intestine, heart, muscle, and brain. Comparable ranking orders were produced by NormFinder. Similar results were obtained at 48 h stimulation. Taken together, these results indicate that B2M and 18S are the most stable gene across tissue types under normal physiological conditions. However, during poly I:C stimulation, no single gene or single pair of genes in the examined set of housekeeping genes can serve as a universal reference across all tissue types. If one gene is preferred, B2M, B2M, UBCE, Actin, B2M/RPL13, B2M, B2M, and RPL13 may be used in spleen, kidney, liver, gill, intestine, brain, muscle, and heart of golden pompano, respectively.
Assuntos
Peixes/genética , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Transcriptoma , Animais , Regulação da Expressão Gênica , RNA Mensageiro/genéticaRESUMO
Background: Because of its strong specificity and high accuracy, real-time quantitative PCR (RT-qPCR) has been a widely used method to study the expression of genes responsive to stress. It is crucial to have a suitable set of reference genes to normalize target gene expression in peanut under different conditions using RT-qPCR. In this study, 11 candidate reference genes were selected and examined under abiotic stresses (drought, salt, heavy metal, and low temperature) and hormone (SA and ABA) conditions as well as across different organ types. Three statistical algorithms (geNorm, NormFinder and BestKeeper) were used to evaluate the expression stabilities of reference genes, and the comprehensive rankings of gene stability were generated. Results: The results indicated that ELF1B and YLS8 were the most stable reference genes under PEG-simulated drought treatment. For high-salt treatment using NaCl, YLS8 and GAPDH were the most stable genes. Under CdCl2 treatment, UBI1 and YLS8 were suitable as stable reference genes. UBI1, ADH3, and ACTIN11 were sufficient for gene expression normalization in low-temperature experiment. All the 11 candidate reference genes showed relatively high stability under hormone treatments. For organs subset, UBI1, GAPDH, and ELF1B showed the maximum stability. UBI1 and ADH3 were the top two genes that could be used reliably in all the stress conditions assessed. Furthermore, the necessity of the reference genes screened was further confirmed by the expression pattern of AnnAhs. Conclusions: The results perfect the selection of stable reference genes for future gene expression studies in peanut and provide a list of reference genes that may be used in the future.
Assuntos
Arachis/genética , Genes de Plantas , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reguladores de Crescimento de Plantas , Padrões de Referência , Seleção Genética , Estresse Fisiológico , Amplificação de Genes , Temperatura Baixa , Perfilação da Expressão Gênica , SecasRESUMO
RT-qPCR is a sensitive and highly efficient technique that is widely used in gene expression analysis and to provide insight into the molecular mechanisms underlying embryonic development. The freshwater prawn, Macrobrachium olfersii is an emerging model organism, but, the stable reference genes of this species need to be identified and validated for RT-qPCR analysis. Thus, the aim of this study was to evaluate the expression stability of six genes (ß-act, GAPDH, EF-1α, RpL8, RpS6, AK) in embryos and in adult tissues (cerebral ganglia, muscle and hepatopancreas) of M. olfersii. The expression stabilities of these genes were evaluated using geNorm, NormFinder, BestKeeper, ΔCt method and integrated tool RefFinder. In the general ranking, RpL8 and RpS6 were the most stable genes in embryos, while RpS6 and RpL8 were the most stable in a combined adult tissue analysis. Analysis of the adult tissues revealed that ß-act and AK were the most stable genes in cerebral ganglia, RpL8 and AK in muscle, and RpS6 and ß-act in hepatopancreas. EF-1α and GAPDH were the least stable genes and as normalizer genes in RT-qPCR affected expression of the Distal-less gene during M. olfersii development. This study provides suitable reference genes for RT-qPCR analysis and allows future studies of the gene expression in M. olfersii for understanding the molecular mechanisms of their development. To our knowledge, this is the first published study that identifies and evaluates reference genes for RT-qPCR analysis in M. olfersii and could be useful as basis for evaluations of reference genes in other prawns.