Reference gene selection for expression studies in the reproductive axis tissues of Magang geese at different reproductive stages under light treatment.
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ABSTRACT:
SUBMITTER: Zhang BB
PROVIDER: S-EPMC8026621 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The molecular mechanism of Chinese cordyceps formation has received a substantial amount of attention because of its usage as traditional Chinese medicine. The formation process of Chinese cordyceps includes two parts: asexual proliferation (Ophiocordyceps sinensis proliferates in the hemolymph of Thitarodes armoricanus larvae) and sexual development (formation and development of fruiting bodies). Therefore, validation of reference genes under different development stages and experimental conditions is crucial for RT-qPCR analysis. However, there is no report on stable reference genes at the development stage of O. sinensis fruiting body. In this study, 10 candidate reference genes, Actin, Cox5, Tef1, Ubi, 18s, Gpd, Rpb1, Try, Tub1 and Tub2, were selected and calculated their expression stability using four methods: geNorm, NormFinder, BestKeeper, and Comparative △Ct. After comprehensive analysis of the results of these four methods with RefFinder, we determined that the most stable reference genes during asexual reproduction of O. sinensis were Tef1 and Tub1, while the most stable reference genes during fruiting body development were Tyr and Cox5, and the most stable reference genes under light-induced conditions were Tyr and Tef1. Our study provides a guidance for reference genes selections at different proliferation processes with light stress of O. sinensis, and represents a foundation for studying the molecular mechanism of Chinese cordyceps formation.
| S-EPMC10118168 | biostudies-literature
Project description:Skeletal muscle is a critical component of goose meat and a significant economic trait of geese. The regulatory roles of miRNAs and lncRNAs in the maturation stage of goose skeletal muscle are still unclear. Therefore, this study conducted experiments on the leg muscles of Magang geese at two stages: 3-day post-hatch (P3) and 3 months (M3). Morphological observations revealed that from P3 to M3, muscle fibers mainly underwent hypertrophy and maturation. The muscle fibers became thicker, nuclear density decreased, and nuclei moved towards the fiber edges. Additionally, this study analyzed the expression profiles of lncRNAs, miRNAs, and mRNAs during the skeletal muscle fiber maturation stage, identifying 1,949 differentially expressed mRNAs (DEMs), 21 differentially expressed miRNAs (DEMIs), and 172 differentially expressed lncRNAs (DELs). Furthermore, we performed enrichment analyses on DEMs, cis-regulatory genes of DELs, and target DEMs of DEMIs, revealing significant enrichment of signaling pathways including MAPK, PPAR, and mTOR signaling pathways. Among these, the MAPK signaling pathway was the only pathway enriched across all three types of differentially expressed RNAs, indicating its potentially more significant role in skeletal muscle maturation. Finally, this study integrated the targeting relationships between DELs, DEMs, and DEMIs from these two stages to construct a ceRNA regulatory network. These findings unveil the potential functions and mechanisms of lncRNAs and miRNAs in the growth and development of goose skeletal muscle and provide valuable references for further exploration of the mechanism underlying the maturation of Magang geese leg muscle.
| S-EPMC10834734 | biostudies-literature
Project description:Amphioxus is a closest living proxy to the ancestor of cephalochordates with vertebrates, and key animal for novel understanding in the evolutionary origin of vertebrate body plan, genome, tissues and immune system. Reliable analyses using quantitative real-time PCR (qRT-PCR) for answering these scientific questions is heavily dependent on reliable reference genes (RGs). In this study, we evaluated stability of thirteen candidate RGs in qRT-PCR for different developmental stages and tissues of amphioxus by four independent (geNorm, NormFinder, BestKeeper and deltaCt) and one comparative algorithms (RefFinder). The results showed that the top two stable RGs were the following: (1) S20 and 18 S in thirteen developmental stages, (2) EF1A and ACT in seven normal tissues, (3) S20 and L13 in both intestine and hepatic caecum challenged with lipopolysaccharide (LPS), and (4) S20 and EF1A in gill challenged with LPS. The expression profiles of two target genes (EYA and HHEX) in thirteen developmental stages were used to confirm the reliability of chosen RGs. This study identified optimal RGs that can be used to accurately measure gene expression under these conditions, which will benefit evolutionary and functional genomics studies in amphioxus.
| S-EPMC5116582 | biostudies-literature
Project description:BACKGROUND: Egg laying in Magang geese is characterized by extended interruption between clutches and lowing laying rate. Both the ovarian follicular development and ovulation characteristics, and the associated endocrine and molecular regulatory mechanisms involved are poorly understood, but could be important for guiding development of molecule aided selection of egg laying performances in geese. This study, therefore, recorded egg-laying characteristics of Magang geese, and the endocrine and molecular regulatory mechanisms of ovarian follicular development, maturation, and ovulation in Magang geese. METHODS: Oviposition, ovarian follicle development, and reproductive hormone and gene expression profiles were observed in a small flock of Magang geese. RESULTS: Greater than 73% of eggs were laid during the day. The average oviposition interval was 46.8 h (36-55 h). It took approximately 18 days for large white follicles to develop into mature F1 follicles; follicular growth was exponential. LHR expression levels increased from the small to the large mature follicles, but FSHR expression decreased in the granulosa and thecal layers. As the follicles matured, inhibin alpha and inhibin betaA expression increased in the granulosa layer. Activin IR, activin IIRA, activin IIRB, and beta-glycan expressions also increased as the follicles increased in size, but were more abundantly expressed in the thecal than in the granulosa layers. During the oviposition cycle, plasma concentrations of gonadal hormones decreased rapidly, whereas the level of PGFM peaked around ovulation. The profiles of activin, inhibin, follistatin, estradiol, and progesterone leading to ovulation were characterized. CONCLUSIONS: The molecular and endocrine mechanisms that regulate follicular development in Magang geese are similar to those in chickens. Moreover, gonadotropin regulation and interaction between activin, inhibin, and follistatin secretion may govern 3-stage maturation in the final preovulatory follicles in Magang geese. The rapid rebound of post-ovulatory secretions of inhibin and follistatin may inhibit recruitment of new SYF recruitment once a sequence of eggs is started, and may limit the egg clutch size to no more than the number of LYFs present before the first sequence egg.
| S-EPMC3716988 | biostudies-literature
Project description:BackgroundApomixis, a natural form of asexual seed production in plants, is considered to have great biotechnological potential for agriculture. It has been hypothesised that de-regulation of the sexual developmental pathway could trigger apomictic reproduction. The genus Boechera represents an interesting model system for understanding apomixis, having both sexual and apomictic genotypes at the diploid level. Quantitative qRT-PCR is the most extensively used method for validating genome-wide gene expression analyses, but in order to obtain reliable results, suitable reference genes are necessary. In this work we have evaluated six potential reference genes isolated from a 454 (FLX) derived cDNA library of Boechera. RNA from live microdissected ovules and anthers at different developmental stages, as well as vegetative tissues of apomictic and sexual Boechera, were used to validate the candidates.ResultsBased on homologies with Arabidopsis, six genes were selected from a 454 cDNA library of Boechera: RPS18 (Ribosomal sub protein 18), Efalpha1 (Elongation factor 1 alpha), ACT 2 (Actin2), UBQ (polyubiquitin), PEX4 (Peroxisomal ubiquitin conjugating enzyme) and At1g09770.1 (Arabidopsis thaliana cell division cycle 5). Total RNA was extracted from 17 different tissues, qRT-PCRs were performed, and raw Ct values were analyzed for primer efficiencies and gene ratios. The geNorm and normFinder applications were used for selecting the most stable genes among all tissues and specific tissue groups (ovule, anthers and vegetative tissues) in both apomictic and sexual plants separately. Our results show that BoechRPS18, BoechEfα1, BoechACT2 and BoechUBQ were the most stable genes. Based on geNorm, the combinations of BoechRPS18 and BoechEfα1 or BoechUBQ and BoechEfα1 were the most stable in the apomictic plant, while BoechRPS18 and BoechACT2 or BoechUBQ and BoechACT2 performed best in the sexual plant. When subgroups of tissue samples were analyzed, different optimal combinations were identified in sexual ovules (BoechUBQ and BoechEfα1), in anthers from both reproductive systems (BoechACT2 and BoechEfα1), in apomictic vegetative tissues (BoechEfα1 and BoechACT2) and sexual vegetative tissues (BoechRPS18 and BoechEfα1). NormFinder ranked BoechACT2 as the most stable in the apomictic plant, while BoechRPS18 was the best in the sexual plant. The subgroups analysis identified the best gene for both apomictic and sexual ovules (BoechRPS18), for anthers from both reproductive system (BoechEfα1) and for apomictic and vegetative tissues (BoechACT2 and BoechRPS18 respectively)ConclusionsFrom a total of six tested genes, BoechRPS18, BoechEfα1, BoechACT2 and BoechUBQ showed the best stability values. We furthermore provide detailed information for the accurate normalization of specific tissue gene expression analyses of apomictic and sexual Boechera.
| S-EPMC3171723 | biostudies-literature
Project description:Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a widely used technique to investigate gene expression levels due to its high throughput, specificity, and sensitivity. An appropriate reference gene is essential for RT-qPCR analysis to obtain accurate and reliable results. To date, no reliable reference gene has been validated for the economically tropical tree, sandalwood (Santalum album L.). In this study, 13 candidate reference genes, including 12 novel putative reference genes selected from a large set of S. album transcriptome data, as well as the currently used ?-actin gene (ACT), were validated in different tissues (stem, leaf, root and callus), as well as callus tissue under salicylic acid (SA), jasmonic acid methyl ester (MeJA), and gibberellin (GA) treatments using geNorm, NormFinder, BestKeeper, Delta Ct and comprehensive RefFinder algorithms. Several novel candidate reference genes were much more stable than the currently used traditional gene ACT. ODD paired with Fbp1 for SA treatment, CSA and Fbp3 for MeJA treatment, PP2C and Fbp2 for GA treatment, as well as Fbp1 combined with Fbp2 for the total of three hormone treatments were the most accurate reference genes, respectively. FAB1A, when combined with PP2C, was identified as the most suitable reference gene combination for the four tissues tested, while the combination of HLMt, PPR and FAB1A were the most optimal reference genes for all of the experimental samples. In addition, to verify our results, the relative expression level of the SaSSy gene was evaluated by the validated reference genes and their combinations in the three S. album tissues and under MeJA treatment. The evaluated reference genes in this study will improve the accuracy of RT-qPCR analysis and will benefit S. album functional genomics studies in different tissues and under hormone stimuli in the future.
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Project description:Training systems generally alter tree architecture, which modulates light microclimate within the canopy, for the purpose of improving photosynthetic efficiency and fruit quality. Gene expression quantification is one of the most important methods for exploring the molecular mechanisms underlying the influence of training systems on pear photosynthesis, and suitable reference genes for gene expression normalization are a prerequisite for this method. In this study, the expression stability of nine common and four novel candidate genes were evaluated in 14 different pear leaf samples in two training systems, including those at four developmental stages (training_period) and from different parts of the trees (training_space), using two distinct algorithms, geNorm and NormFinder. Our results revealed that SKD1 (Suppressor of K+ Transport Growth Defect1)/ YLS8 (Yellow Leaf Specific 8) and ARM (Armadillo) were the most stable single reference genes for the 'training_period' and 'training_space' subsets, respectively, although these single genes were not as stable as the optimal pairs of reference genes, SKD1+YLS8 and ARM+YLS8, respectively. Furthermore, the expression levels of the PpsAPX (Ascorbate peroxidase) gene showed that the arbitrary use of reference genes without previous testing could lead to misinterpretation of data. This work constitutes the first systematic analysis regarding the selection of superior reference genes in training system studies, facilitating the elucidation of gene function in pear and providing valuable information for similar studies in other higher plants.
| S-EPMC6107188 | biostudies-literature
Project description:In biological research the analysis of gene expression levels in cells and tissues can be a powerful tool to gain insights into biological processes. For this, quantitative RT-PCR (RT-qPCR) is a popular method that often involve the use of constitutively expressed endogenous reference (or 'housekeeping') gene for normalization of data. Thus, it is essential to use reference genes that have been verified to be stably expressed within the specific experimental setting. Here, we have analysed the expression stability of 12 commonly used reference genes (Actb, B2m, Gapdh, Hprt, Pgk1, Rn18s, Rpl13a, Rps18, Rps29, Sdha, Tbp and Ubc) across several juvenile and adult rat tissues (liver, adrenal, prostate, fat pad, testis and ovaries), both under normal conditions and following exposure to various chemicals during development. Employing NormFinder and BestKeeper softwares, we found Hprt and Sdha to be amongst the most stable genes across normal and manipulated tissues, with several others also being suitable for most tissues. Tbp and B2m displayed highest variability in transcript levels between tissues and developmental stages. It was also observed that the reference genes were most unstable in liver and testis following toxicological exposure. For future studies, we propose the use of more than one verified reference gene and the continuous monitoring of their suitability under various experimental conditions, including toxicological studies, based on changes in threshold (Ct) values from cDNA samples having been reverse-transcribed from a constant input concentration of RNA.
| S-EPMC4375968 | biostudies-literature
Project description:Chrysoperla nipponensis (Okamoto), which has the unique diapause phenotype distinguishable from nondiapause adult, is an ideal model organism for studying the mechanism of reproductive diapause. However, there is no reliable and effective reference genes used for the reproductive diapause study of C. nipponensis. Therefore, in this study, we evaluated the expression stability of 10 candidate reference genes (Tub1, Arpc5, EF1a, 128up, RpS5, RpS26e, GAPDH, Arp3, Actin, α-Tub) in adults under diapause and nondiapause induction conditions using four statistical algorithms including GeNorm, NormFinder, Bestkeeper, and ∆CT method. Results showed that Arp3 and Tub1 were the most stable reference genes in all samples and in the adult tissues group. Arp3 and RpS5 were the most stable reference genes in the development degree group. α-Tub and EF1a were unstable reference genes under the conditions of this study. Meanwhile, to verify the reliability of the reference genes, we evaluated the relative expression levels of Vg and VgR in different treatments. Significant upregulation and downregulation in expression level of two genes in response to diapause termination and diapause fat body tissue was, respectively, observed when using Arp3 as the reference gene but not when using an unstable reference gene. The reference genes identified in this work provided not only the basis for future functional genomics research in diapause of C. nipponensis and will also identify reliable normalization factors for real-time quantitative real-time polymerase chain reaction data for other related insects.
| S-EPMC7433768 | biostudies-literature
Project description:Okra (Abelmoschus esculentus L.) is a particular vegetable with both edible and medicinal values. However, the expression pattern of the okra reference genes in response to abiotic stress has not been explored. In the present study, 18 potential reference genes were selected from okra in various tissues and abiotic stress conditions, and their expression levels were detected by Real-Time quantitative PCR (RT-qPCR). Their expression stabilities were calculated by four algorithms (geNorm, NormFinder, BestKeeper, and RefFinder). Under cold stress, the most stable genes included GAPC1 and CYP (leaf), CYP and ACT7 (root), HIS6 and GAPC1 (stem), and HIS6 and 60s (different tissues). Under salt stress, EF-1α and UBQ (leaf), EF-1α and UBQ (root), TUA4 and Eif (stem), and HIS6 and Eif (different tissues) were the most stable genes. Under drought stress, UBQ and Eif in the leaf, HIS6 and Eif in the root, TUA4 and HIS6 in the stem, and UBQ and Eif in different tissues were most stably expressed in okra. In addition, complete sequencing results by RefFinder showed that HIS6 and ACT7 in the leaf, HIS6 and Eif in the root, UBC5B and 60s in the stem, and HIS6 and Eif in different tissues, were most the suitable reference genes for okra. Furthermore, AeMYB1R1 transcription factor was used to verify the reliability of RT-qPCR values. In summary, this study was carried out to demonstrate the potential reference genes of okra under abiotic stress, aiming to provide a molecular basis for functional gene analysis and regulatory mechanism research of okra.
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