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Freshly collected seeds of Amomum tsaoko demonstrate obvious dormancy. Therefore, the selection of stable reference genes during seed dormancy release is very important for the subsequent functional research of related genes. In this study, ten commonly used reference genes(GAPDH, 40S, actin, tubulin, EIF4A-9, EIF2α, UBC, UBCE2, 60S, and UBQ) were selected as candidates for quantitative Real-time polymerase chain reaction(qRT-PCR) of the embryo samples of A. tsaoko at different dormancy release stages. Three kinds of software(BestKeeper, geNorm, and Normfinder) and the Delta CT method were used to evaluate the expression stability of the candidate reference genes, and the RefFinder online tool was employed to integrate the results and generate a comprehensive ranking. The results showed that the expression levels of the ten candidate reference genes differed greatly in different embryo samples. GAPDH and UBC had high expression levels, as manifested by the small Ct values. GeNorm identified 40S and UBCE2 as the most stable genes. NormFinder ranked EIF2α as the most stable gene and UBC as the least stable gene. UBCE2 was found to be the most stable gene and actin the least stable one by BestKeeper. Delta CT analysis suggested that the expression of 40S was most stable. UBCE2 was recommended as the most stably expressed gene by RefFinder. Thus, UBCE2 is the ideal reference gene for qRT-PCR analysis of A. tsaoko seeds at different dormancy release stages. The results may lay a foundation for analyzing the expression of related genes during seed dormancy release of A. tsaoko.
Subject(s)
Amomum , Gene Expression Profiling , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Seeds/geneticsABSTRACT
Objective: To clone and screen the stable internal reference genes from Dipsacus asper for qRT-PCR analysis correction, so as to provide a preliminary basis for future research on expression analysis and regulation mechanism of D. asper functional genes. Methods: The internal reference genes of Actin, Tubulin and GAPDH gene families were screened and cloned from D. asper transcriptome database. The D. asper plants from different origins, different tissues and different developmental stages were used to obtain expression information of each gene by qRT-PCR. The expression stability of each gene was analyzed by geNorm, NormFinder, BestKeeper, Delta CT and RefFinder, and the best genes were synthetically evaluated and screened. Results: Ten core fragments for candidate internal reference genes were cloned, belonging to three gene families: Actin, Tubulin and GAPDH, with high homology among them. The results of stability analysis showed that the expression of DaACT103 was stable and relatively high in different regions and tissues, while the expression of DaTUB5 was stable and relatively low in different developmental stages. Conclusion:s DaACT103 and DaTUB5 are suitable as the internal reference genes for D. asper. DaACT103 is used as the internal reference gene with high abundances and DaACT105 is used as the internal reference gene with low abundances.
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Abstract Rabbit with hypercholesterolaemia is an important model for studying cholesterol metabolism disease. This study aimed to evaluate the expression stability of nine reference genes for quantitative PCR (qPCR) analysis in adrenal gland, liver, spleen, and kidney tissue from rabbits with hypercholesterolaemia. In total, 30 male Harbin Large White (HLW) rabbits were fed a normal feed (n = 15) or a high cholesterol feed (n = 15) for 8 weeks to induce hypercholesterolaemia. Nine reference genes were verified by qPCR using cDNA extracted from rabbit tissue samples. For qPCR analysis, reference genes were evaluated using the RefFinder and GeNorm algorithms. Overall, seven rabbits with hypercholesterolaemia were identified based on body weight and total cholesterol measurements. Combining the results of the RefFinder and GeNorm algorithms, the most stable reference genes were hypoxanthine phosphoribosyltransferase 1 (Hprt1) and eukaryotic translation elongation factor 1 alpha 1 (Eef1a1) in the adrenal gland, β-2-microglobulin (B2m) and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) in the liver, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (Ywhaz) and Gapdh in the spleen, and peptidylprolyl isomerase (Ppia), β-actin (Actb), succinate dehydrogenase complex subunit A flavoprotein (Sdha), and B2m in the kidney. Taken together, our results confirmed that Hprt1 and Eef1a1, B2m and Gapdh, Ywhaz and Gapdh, and Ppia, Actb, Sdha, and B2m were the best reference genes for qPCR analyses in adrenal gland, liver, spleen, and kidney tissue, respectively, of rabbits with hypercholesterolaemia.
Subject(s)
Animals , Rabbits , Eukaryotic Initiation Factor-1 , Adrenal Glands , Real-Time Polymerase Chain Reaction/instrumentation , Hypercholesterolemia/chemically induced , Hypoxanthine Phosphoribosyltransferase/analysisABSTRACT
The development and metabolism of medicinal plant is affected by many factors, among which the effect from endophytic fungi has been noticed recently and has become one of hot fields. In order to explore the effect of endophytic fungi on gene expression in R. crenulata, RNA-sequencing was used to find genes involved in metabolic pathways, and the differential genes were verified by real-time fluorescent quantitative PCR. The method of 2-△△Ct was used to analyze the relative expression levels of genes in related metabolic pathways, which was used to verify the result of transcriptomics sequencing. The results showed that the endophytic fungus, P. fortinii, could up-regulate the gene expression in lipid metabolic pathway of R. crenulata. In signal transduction pathway, the genes were influenced at different level but the gene expressions were significantly increased under control of Notch signaling pathway, which was 34 times of that in control. The gene expressions of environmental adaption pathway were up-regulated in R. crenulata after inoculation of P. fortinii. This study could provide help for further understanding on mechanism of plant-fungus interaction, root cause of geoherbalism of medicinal plant and exploring bio-function of endophytic fungi.
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Objective: To screen reference genes for real time quantitative PCR (qRT-PCR) research in Ampelopsis grossedentata. Methods: On the basis of the conserved sequences among plant species, six candidate reference genes (including Actin, 18 S-rRNA, GAPDH, α-Tubulin, β-Tubulin, and UBQ) were cloned from A. grossedentata by RT-PCR in this study. The expression stability of each reference gene in different tissues (shoot tip, young leaf, mature leaf, old leaf, stem, and root) were analyzed by three softwares (GeNorm, NormFinder, and BestKeeper), followed by validation of the expression pattern of AgPAL by qRT-PCR. Results: Actin, 18 S-rRNA, and GAPDH expressed most stably in all samples and were suitable for reference genes, which were further confirmed by the transcript level analysis result of AgPAL in different tissues. Conclusion: This is the first report on the screening and validation of reference genes for qRT-PCR in A. grossedentata, which benefits future studies on gene expression in this species.
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Objective To identify stable reference genes in different growth stages and different organs of Bupleurum chinese Methods All Ct values of 18 candidate internal reference genes were obtained by real-time quantitative PCR. Three software (Bestkeeper, NormFinder, and GeNorm) based on different algorithms were used to analyze the stability of the internal reference gene. The Pearson correlation coefficient was also analyzed. Results The Ct values of all candidate genes were relatively broad. ADF1b, ADF5, ADF7, eIF2b, and ACT2 were the most stable reference genes, whereas the gene of eIF6 was the least stable of the reference gene. The results of three softwares showed significant correlation. Conclusion Real-time fluorescence quantitative PCR combined with three different algorithms for the screening and validation of the reference gene of B. chinese is feasible. The homogenization of the target gene by the reference genes of the present study is helpful to improve the accuracy and reliability of gene expression analysis in molecular genetic research of B. chinese.
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Objective To identify reliable reference genes (RGs) for normalization of real-time PCR (RT-qPCR) data in Codonopsis pilosula. Methods The expression profiles of 10 candidate RGs (GAPDH, ACT, α-TUB, β-TUB, UBQ, CYP, EF-1α, NAC, F-box and PP2A) were examined in C. pilosula during the phenological period. The raw materials examined included roots, stems, leaves, and flower buds at flowering and boll-forming stages, five growth stages of untreated and treated roots with plant growth retardant. The best-suited RGs were accessed using geNorm, NormFinder, BestKeeper, and RefFinder algorithms. Results The best-ranked references genes differed across the samples. GAPDH and PP2A were the most suitable for expression analysis in untreated tissues while GAPDH, α-TUB, and PP2A were ranked as the three most stably expressed genes in untreated roots, while NAC and CYP were the most stably expressed genes in stressed (i.e., treated) roots. The expression of UGPase, a key enzyme for CPP biosynthesis, was determined to further validate the selected RGs. Conclusion A total of 10 RGs can be used as reference genes of C. pilosula, however the appropriate one should be used as it may chance.
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Abstract The selection of suitable reference genes is crucial for accurate quantification of gene expression and can add to our understanding of host–pathogen interactions. To identify suitable reference genes in Pandora neoaphidis, an obligate aphid pathogenic fungus, the expression of three traditional candidate genes including 18S rRNA(18S), 28S rRNA(28S) and elongation factor 1 alpha-like protein (EF1), were measured by quantitative polymerase chain reaction at different developmental stages (conidia, conidia with germ tubes, short hyphae and elongated hyphae), and under different nutritional conditions. We calculated the expression stability of candidate reference genes using four algorithms including geNorm, NormFinder, BestKeeper and Delta Ct. The analysis results revealed that the comprehensive ranking of candidate reference genes from the most stable to the least stable was 18S (1.189), 28S (1.414) and EF1 (3). The 18S was, therefore, the most suitable reference gene for real-time RT-PCR analysis of gene expression under all conditions. These results will support further studies on gene expression in P. neoaphidis.
Subject(s)
Entomophthorales/genetics , Genes, Fungal , Gene Expression Profiling/methods , Gene Expression Profiling/standards , Reference Standards , Real-Time Polymerase Chain Reaction/methods , Real-Time Polymerase Chain Reaction/standards , Peptide Elongation Factor 1/genetics , /genetics , /geneticsABSTRACT
Jatropha curcas L represent a potential source of raw material for the production of biodiesel. The aim this study was to find potential candidate reference genes in J. curcas tissues. Three softwares were utilized to verify which would be the most stable reference genes in qPCR assay: GeNorm, NormFinder and BestKeeper. The most stable reference genes in developing J. curcas seeds suggested by GeNorm were GAPDH, UCP, actin. However, the best combinations of stable genes in each tissue were identified separately under stress conditions: EF1-α, PP2A2 and GAPDH in total stress, however, in SA stress, four genes were required for normalization: PP2A2, EF1-α, GAPDH and PUB. In PEG stress, four genes also were required: PP2A2, EF1-α, GAPDH and PUB, while in NaCl stress, five genes were necessary: PP2A2, GAPDH, EF1-α, PUB and Tβ2. These results are in accordance with two other programs used in this study (NormFinder, BestKeeper). In addition, the transcript levels of Jc-SRG-2 seem to be more correlated with stress responses than changes in transcript levels of Jc-SRG-1, mainly of leaves in exposure to 3-12 h on PEG and NaCl stress. Taken together, GAPDH and PP2A2 were regarded as being the best reference to provide guidelines for the selection of potential references genes under these study conditions.
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Objective To identify the reliable reference genes for gene expression analysis of the pericarp and seed of Amomum villosum Lour. by using real-time fluorescence quantitative polymerase chain reaction ( qRT-PCR). Methods Using the fruits ( separated into peels and seeds) of A. villosum at three different developmental periods as the experimental material, 5 candidate reference genes (β-actin, EF-1α, GAPDH, PGK, TUA) with steady expression were screened out by the high throughout sequencing of transcriptome and expression profile data. The qRT-PCR technique was applied to study the expression levels of 5 candidate reference genes in different samples. The stability of the candidate reference genes were evaluated by GeNorm and NormFinder software. Results The 5 reference genes had different stabilities in the pericarp and seed of A. villosum Lour. at different development periods . The order of the steadiness of reference genes showed by GeNorm was EF-1α = TUA>PGK>GAPDH>β-actin. The results of NormFinder revealed that EF-1α was the most stable, followed by TUA, and the order of the other three genes was as same as the results of GeNorm. Conclusion EF-1αand TUA could be used as double reference genes for the normalization of gene expression in A. villosum fruits at different developmental periods by using qRT-PCR.
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The aim of this review was to evaluate the importance of the real-time PCR (qRT-PCR) as a technique for mRNA expression analysis in different tissues. Real-time PCR is widely used for quantification of mRNA levels and is a fundamental tool for basic research, molecular medicine and biotechnology.Genes of references are expressed in a wide variety of tissues and cells with minimal variations in their expression levels, and thus are used to normalize data of mRNA quantification. Software programs, such as geNorm, BestKeeper and NormFinder, have been developed to perform the normalization of data, which help to choose the most stable reference gene. Several genes, such as GAPDH, β-actin, β-tubulin, PGK, UBQ, RPL-19 and 18S rRNA have been suggested as standards in PCR studies, but these genes can have variation in their expression in different tissues, reinforcing the idea that there is no ideal reference gene.
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Objective To investigate the proper inner control genes suitable for mRNA expression level comparison of aging rat tissues.Methods Real-time reverse transcription PCR was used to examine in aging rat tissues the expression level of G3pd(glyceraldehyde-3-phosphate dehydrogenase),ACTB(?-actin),H3f3b(H3 histone,family 3B),Arbp(acidic ribosomal phosphoprotein P0)and 18S(18S ribosomal RNA).Results The most stably expressed housekeeping gene in aging rat kidney was ACTB,in heart and lung G3pd showed the minimum variation;Arbp expression was the most stable one in different tissues.Conclusion For aging rat intra-tissue mRNA normalization at least two housekeeping genes should be used: one is the ribosomal RNA gene 18S and another one is Arbp.