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1.
Zhao W Li Y Gao P Sun Z Sun T Zhang H 《Journal of industrial microbiology & biotechnology》2011,38(9):1279-1286
Lactobacillus casei Zhang, a potential probiotic strain isolated from homemade koumiss in Inner Mongolia of China, has been sequenced and deposited
in GenBank. Real-time quantitative PCR is one of the most widely used methods to study related gene expression levels of Lactobacillus casei Zhang. For accurate and reliable gene expression analysis, normalization of gene expression data using one or more appropriate
reference genes is essential. We used three statistical methods (geNorm, NormFinder, and BestKeeper) to evaluate the expression
levels of five candidate reference genes (GAPD, gyrB, LDH, 16s rRNA, and recA) under different culture conditions and different growth phases to find a suitable housekeeping gene which can be used as
internal standard. The results showed that the best reference gene was GAPD, and a set of two genes, GAPD and gyrB (which were the most stable reference genes), is recommended for normalization of real-time quantitative PCR experiments
under all the different experimental conditions tested. The systematic validation of candidate reference genes is important
for obtaining reliable analysis results of real-time quantitative PCR studies in gene expression levels of Lactobacillus casei Zhang. 相似文献
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Identification of suitable reference genes for quantitative real‐time PCR normalization in blotched snakehead Channa maculata
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H. Mao K. Chen X. Zhu Q. Luo J. Zhao W. Li X. Wu H. Xu 《Journal of fish biology》2017,90(6):2312-2322
A systematic study was conducted to identify reliable reference genes for normalization of gene expression analysis in the blotched snakehead Channa maculata under normal physiological conditions. Firstly, the partial complementary (c)DNA of nine candidate reference genes (actb, tmem104, ube2l3, ef1α, churc1, tmem256, rpl13a, sep15 and g6pd) were cloned from C. maculata. The expression levels of these genes were then assessed in embryos of different developmental stages and various tissue types of adult fish using quantitative real‐time (qrt‐)PCR. RefFinder algorithm was used to evaluate the expression stability of these genes based on their cycle‐threshold (Ct) values in the qrt‐PCR analysis. Results showed that there was no single best reference gene for all stages of embryos and adult tissues tested. Furthermore, it was found that, among the nine candidate genes tested, actb and tmem104 were the most stable reference genes across adult tissue types, while sep15 and tmem256 were the most stable ones across developmental stages of embryos. These stable reference genes are recommended for normalization of gene expression analysis in C. maculata. 相似文献
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Zhong Q Zhang Q Wang Z Qi J Chen Y Li S Sun Y Li C Lan X 《Marine biotechnology (New York, N.Y.)》2008,10(3):310-318
Differential expression of genes is crucial to embryogenesis. The analysis of gene expression requires appropriate references
that should be minimally regulated during the embryonic development. To select the most stable genes for gene normalization,
the expression profiles of eight commonly used reference genes (ACTB, GAPDH, rpL17, α-Tub, EF1-α, UbcE, B2M, and 18S rRNA)
were examined during Japanese flounder (Paralichthys olivaceus) embryonic development using quantitative real-time polymerase chain reaction. It was found that all seven mRNA genes appeared
to be developmentally regulated and exhibited significant variation of expression. However, further analyses revealed the
stage-specific expression stability. Hence when normalization using these mRNA genes, the differential and stage-related expression
should be considered. 18S rRNA gene, on the other hand, showed the most stable expression and could be recommended as a suitable
reference gene during all embryonic developmental stages in P. olivaceus. In summary, our results provided not only the appropriate reference gene for embryonic development research in P. olivaceus, but also possible guidance to reference gene selection for embryonic gene expression analyses in other fish species. 相似文献
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The identification of suitable reference genes is critical for obtaining reliable results from gene expression studies using quantitative real-time PCR (qPCR) because the expression of reference genes may vary considerably under different experimental conditions. In most cases, however, commonly used reference genes are employed in data normalization without proper validation, which may lead to incorrect data interpretation. Here, we aim to select a set of optimal reference genes for the accurate normalization of gene expression associated with intramuscular fat (IMF) deposition during development. In the present study, eight reference genes (PPIB, HMBS, RPLP0, B2M, YWHAZ, 18S, GAPDH and ACTB) were evaluated by three different algorithms (geNorm, NormFinder and BestKeeper) in two types of muscle tissues (longissimus dorsi muscle and biceps femoris muscle) across different developmental stages. All three algorithms gave similar results. PPIB and HMBS were identified as the most stable reference genes, while the commonly used reference genes 18S and GAPDH were the most variably expressed, with expression varying dramatically across different developmental stages. Furthermore, to reveal the crucial role of appropriate reference genes in obtaining a reliable result, analysis of PPARG expression was performed by normalization to the most and the least stable reference genes. The relative expression levels of PPARG normalized to the most stable reference genes greatly differed from those normalized to the least stable one. Therefore, evaluation of reference genes must be performed for a given experimental condition before the reference genes are used. PPIB and HMBS are the optimal reference genes for analysis of gene expression associated with IMF deposition in skeletal muscle during development. 相似文献
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Karin Mitter Georgios Kotoulas Antonios Magoulas Victor Mulero Pilar Sepulcre Antonio Figueras Beatrice Novoa Elena Sarropoulou 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2009,153(4):340-347
The expression level of mRNA can vary significantly in different experimental conditions, such as stress, infection, developmental stage or tissue. Suitable reference genes are expected to exhibit constant expression levels. However no single gene is constitutively expressed in all cell types and under all experimental conditions. It has become clear that expression stability of the intended reference gene has to be examined before each experiment. For expression studies using quantitative real-time PCR (qPCR) at least two reference genes have to be applied. So far expression studies in the European seabass (Dicentrarchus labrax) as well as in the Gilthead seabream (Sparus aurata) have been performed with only one reference gene (S18, Ef-1 alpha or Gapdh). Though significant variations showed up in other teleost species such as the Atlantic halibut and the zebrafish affirming the need for proper normalization strategies, the present study aims at identifying suitable reference genes among nine candidates [glyceraldehyde-phosphate-dehydrogenase (Gapdh), β-actin (two regions of β-actin), 40S ribosomal protein S30 (Fau), ribosomal protein L13 a (L13a), β2-tubulin (Tubb2) and tyrosine 3 monooxygenase/tryptophan 5-monooxygenase activation protein (Tyr)] for expression analysis of 8 developmental stages and a tissue panel (spleen, liver, kidney and brain) with samples infected with Nodavirus and Vibrio anguillarum in D. labrax. Besides the analysis of raw Ct-values, the gene expression stability was determined using two different software applications BestKeeper and NormFinder. According to both algorithms the best two reference genes for an appropriate normalization approach during D. labrax development are Ef-1 alpha and L13a whereas in the tissue panel Fau and L13a are recommended for qPCR normalization. 相似文献
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The validation of housekeeping genes (HKGs) for normalization of RNA expression in Real-Time PCR is crucial to obtain the most reliable results. There is limited information on reference genes used in the study of gene expression in milk somatic cells and the frozen whole blood of goats. Thus, the aim of this study was to propose the most stable housekeeping genes that can be used as a reference in Real-Time PCR analysis of milk somatic cells and whole blood of goats infected with caprine arthritis encephalitis virus (CAEV). Animals were divided into two groups: non-infected (N = 13) and infected with CAEV (N = 13). Biological material (milk somatic cells and whole blood) was collected 4 times during the lactation period (7, 30, 100 and 240 days post-partum). The expression levels of candidate reference genes were analyzed using geNorm and NormFinder software. The stability of candidates for reference gene expression was analyzed for CAEV-free (control) and CAEV-infected groups, and also for both groups together (combined group). The stability of expression of β-actin (ACTB), glyceraldehyde-3P-dehydrogenase (GAPDH), cyclophilin A (PPIA), RNA18S1, ubiquilin (UBQLN1) and ribosomal protein large subunit P0 (RPLP0) was determined in milk somatic cells, while ACTB, PPIA, RPLP0, succinate dehydrogenase complex subunit A (SDHA), zeta polypeptide (YWHAZ), battenin (CLN3), eukaryotic translation initiation factor 3K (EIF3K) and TATA box-binding protein (TBP) were measured in frozen whole blood of goats. PPIA and RPLP0 were considered as the most suitable internal controls as they were stably expressed in milk somatic cells regardless of disease status, according to NormFinder software. Furthermore, geNorm results indicated the expression of PPIA/RPLP0 genes as the best combination under these experimental conditions. The results of frozen whole blood analysis using NormFinder software revealed that the most stable reference gene in control, CAEV-infected and combined groups is YWHAZ, and – according to the geNorm results – the combined expression of PPM/YWHAZ genes is the best reference in the presented experiment. The usefulness in gene expression analysis of whole blood samples frozen immediately in liquid nitrogen and stored at -80 °C was also proved. 相似文献
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Patricia Castro Belén Román Josefa Rubio José V. Die 《Molecular breeding : new strategies in plant improvement》2012,29(1):261-274
Normalisation to a reference gene is the most common method of internally controlling for error in quantitative PCR (qPCR)
experiments. Studies based on qPCR in chickpea have been carried out using potential reference genes exclusively. Inappropriate
normalisation may result in the acquisition of biologically irrelevant data. We have tested the expression of 12 candidate
internal control genes in 36 samples representing different organs/developmental stages, genotypes and stress conditions.
The most stably expressed genes were PUBQ, GAPDH, UBQ and bHLH, whereas 18S rRNA and EF-1a showed considerable regulation. The most suitable combination of reference genes for the particular experimental sets tested
is provided. To illustrate the use of chickpea reference genes, we checked the expression of a putative defence gene in two
different genotypes infected with Ascochyta rabiei (Pass.) Lab. The set of reference genes presented here will enable the more accurate and reliable normalisation of qPCR results
for gene expression studies in this important legume crop. Our findings can be used as a starting point for reference gene
selection in experimental conditions different from those tested here. 相似文献
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Reference genes for RT‐qPCR normalisation in different tissues,developmental stages and stress conditions of amaranth
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F. P. Vera Hernández M. Martínez Núñez M. Ruiz Rivas R. E. Vázquez Portillo M. D. Bibbins Martínez S. Luna Suárez F.de F. Rosas Cárdenas 《Plant biology (Stuttgart, Germany)》2018,20(4):713-721
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Zhi‐Wei Kang Fang‐Hua Liu Hong‐Gang Tian Meng Zhang Shan‐Shan Guo Tong‐Xian Liu 《Insect Science》2017,24(2):222-234
The green peach aphid, Myzus persicae Sulzer (Hemiptera, Aphididae), is an important cosmopolitan pest. Real time qRT‐PCR has been used for target gene expression analysis on M. persicae. Using real time qRT‐PCR, the expression levels are normalized on the basis of the reliable reference genes. However, to date, the stability of available reference genes has been insufficient. In this study, we evaluated nine candidate reference genes from M. persicae under diverse experimental conditions. The tested candidate genes were comprehensively ranked based on five alternative methods (RefFinder, geNorm, Normfinder, BestKeeper and the comparative ΔCt method). 18s, Actin and ribosomal protein L27 (L27) were recommended as the most stable reference genes for M. persicae, whereas ribosomal protein L27 (L27) was found to be the least stable reference genes for abiotic studies (photoperiod, temperature and insecticide susceptibility). Our finding not only sheds light on establishing an accurate and reliable normalization of real time qRT‐PCR data in M. persicae but also lays a solid foundation for further studies of M. persicae involving RNA interference and functional gene research. 相似文献
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Yinjie Wang Yongxia Zhang Qingquan Liu Liangqin Liu Suzhen Huang Haiyan Yuan 《Phyton》2021,90(1):277-290
Quantitative real-time PCR (qPCR) is an effective and widely used
method to analyze expression patterns of target genes. Selection of stable reference genes is a prerequisite for accurate normalization of target gene expression
by qRT-PCR. In Iris germanica L., no studies have yet been published regarding
the evaluation of potential reference genes. In this study, nine candidate reference
genes were assessed at different flower developmental stages and in different tissues by four different algorithms (GeNorm, NormFinder, BestKeeper, and RefFinder). The results revealed that ACT11 (Actin 11) and EF1α (Elongation factor
1 alpha) were the most stable reference genes in different tissues, whereas TUA
(Tubulin alpha) and UBC9 (Ubiquitin-protein ligase 9) were the most stable ones
in different flower developmental stages. UBC9 and ACT11 were the most stable
reference genes in all of the tested samples, while the SAMDC (S-Adenosylmethionine decarboxylase) showed the least stability. Finally, to validate the suitability of the selected reference genes, the relative expression level of IgTPS
(beta-caryophyllene synthase) was assessed and highlighted the importance of
suitable reference gene selection. This work constitutes the first systematic evaluation of potential reference genes in I. germanica and provides guidelines for
future research on gene function and molecular mechanisms on I. germanica
and related species. 相似文献
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