scholarly journals Application of the common base method to regression and analysis of covariance (ANCOVA) in qPCR experiments and subsequent relative expression calculation

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Michael T. Ganger ◽  
Geoffrey D. Dietz ◽  
Patrick Headley ◽  
Sarah J. Ewing
Keyword(s):  
Gene Expression ◽  
Quantitative Polymerase Chain Reaction ◽  
Analysis Of Covariance ◽  
Relative Expression ◽  
Common Base ◽  
Linear Relationships ◽  
Base Method ◽  
Independent Variable ◽  
The Common

Abstract Background Quantitative polymerase chain reaction (qPCR) is the technique of choice for quantifying gene expression. While the technique itself is well established, approaches for the analysis of qPCR data continue to improve. Results Here we expand on the common base method to develop procedures for testing linear relationships between gene expression and either a measured dependent variable, independent variable, or expression of another gene. We further develop functions relating variables to a relative expression value and develop calculations for determination of associated confidence intervals. Conclusions Traditional qPCR analysis methods typically rely on paired designs. The common base method does not require such pairing of samples. It is therefore applicable to other designs within the general linear model such as linear regression and analysis of covariance. The methodology presented here is also simple enough to be performed using basic spreadsheet software.

scholarly journals The Selection of Reference Genes for Quantitative Real-Time PCR in the Ashidan Yak Mammary Gland During Lactation and Dry Period

Animals ◽  
2019 ◽  
Vol 9 (11) ◽  
pp. 943 ◽  
Author(s):  
Xiaoyun Wu ◽  
Xuelan Zhou ◽  
Xuezhi Ding ◽  
Min Chu ◽  
Chunnian Liang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammary Gland ◽  
Real Time ◽  
Milk Yield ◽  
Reference Genes ◽  
Quantitative Polymerase Chain Reaction ◽  
Expression Stability ◽  
Dry Period ◽  
Relative Expression ◽  
Selection Of

Investigating the critical genes related to milk synthesis is essential for the improvement of the milk yield of the yak. Real-time quantitative polymerase chain reaction (RT-qPCR) is a reliable and widely used method to measure and evaluate gene expression levels. Selection of suitable reference genes is mandatory to acquire accurate normalization of gene expression results from RT-qPCR. To select the most stable reference genes for reliable normalization of mRNA expression by RT-qPCR in the mammary gland of the Ashidan yak, we selected 16 candidate reference genes and analyzed their expression stability at different physiological stages (lactation and dry period). The expression stability of the candidate reference genes was assessed using geNorm, NormFinder, BestKeeper, Delta Ct, and RefFinder methods. The results showed that the hydroxymethylbilane synthase gene (HMBS) and the tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide gene (YWHAZ) were the most stable genes across all treatment samples. The reliability of selected reference genes was validated by normalizing relative expression of the lactation-related 60S ribosomal protein L35 gene (RPL35). The relative expression of RPL35 varied considerably according to the different reference genes. This work provides valuable information to further promote research in the molecular mechanisms involved in lactation and mammary gland development and provides a foundation for the improvement of the milk yield and quality of the Ashidan yak.

scholarly journals A sensitive spectrophotometric method for the determination of H2-receptor antagonists by means of N-bromosuccinimide and p-aminophenol

2008 ◽  
Vol 58 (1) ◽  
pp. 87-97 ◽  
Author(s):  
Ibrahim Darwish ◽  
Samiha Hussein ◽  
Ashraf Mahmoud ◽  
Ahmed Hassan
Keyword(s):  
Spectrophotometric Method ◽  
Correlation Coefficients ◽  
Receptor Antagonists ◽  
Pharmaceutical Dosage Forms ◽  
Linear Relationships ◽  
Colored Product ◽  
Subsequent Measurement ◽  
The Common ◽  
Sensitive Spectrophotometric Method

A sensitive spectrophotometric method for the determination of H2-receptor antagonists by means ofN-bromosuccinimide andp-aminophenolA simple, accurate and sensitive spectrophotometric method for determination of H2-receptor antagonists: cimetidine (CIM), famotidine (FAM), nizatidine (NIZ), and ranitidine hydrochloride (RAN) has been fully developed and validated. The method was based on the reaction of these drugs with NBS and subsequent measurement of the excessN-bromosuccinimide by its reaction withp-aminophenol to give a violet colored product (λmaxat 552 nm). Decrease in the absorption intensity (ΔA) of the colored product, due to the presence of the drug, was correlated with its concentration in the sample solution. Different variables affecting the reaction were carefully studied and optimized. Under optimal conditions, linear relationships with good correlation coefficients (0.9988--0.9998) were found between ΔAvalues and the corresponding concentrations of the drugs in a concentration range of 8--30, 6--22, 6--25, and 4--20 μg mL-1for CIM, FAM, NIZ, and RAN, respectively. Limits of detection were 1.22, 1.01, 1.08, and 0.74 μg mL-1for CIM, FAM, NIZ, and RAN, respectively. The method was validated in terms of accuracy, precision, ruggedness, and robustness; the results were satisfactory. The proposed method was successfully applied to the analysis of the above mentioned drugs in bulk substance and in pharmaceutical dosage forms; percent recoveries ranged from 98.5 ± 0.9 to 102.4 ± 0.8% without interference from the common excipients. The results obtained by the proposed method were comparable with those obtained by the official methods.

scholarly journals Identification of reliable reference genes for gene expression studies in maternal reproductive tissues and fetal tissues of pregnant cows

2020 ◽  
Author(s):  
Lei Cheng ◽  
Jie Yu ◽  
Xiuzhong Hu ◽  
Min Xiang ◽  
Yu Xia ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Target Genes ◽  
Quantitative Polymerase Chain Reaction ◽  
Related Factor ◽  
Expression Stability ◽  
Relative Expression ◽  
Reproductive Tissues ◽  
Fetal Tissues ◽  
Selection Of

Abstract Background: The relationship between the conceptus and the maternal uterine environment is crucial for the successful establishment and maintenance of pregnancy in cattle. Gene expression analysis of the conceptus and maternal reproductive tissues is a favorable method to assess the embryonic maternal interaction. The reliability of the commonly used method reverse transcription-quantitative polymerase chain reaction (RT-qPCR) depends on proper normalization to stable reference genes (RGs). The objective of this study was to determine the expression stability of ten potential RGs (SUZ12, CNOT11, ACTB, RPL19, RPS9, GAPDH, TBP, HPRT1, SDHA and PPIA) in maternal reproductive tissues and fetal tissues, and to analyze the effect of RG selection on the calculation of the relative expression of target genes. Results: The expression stability of ten potential RGs was analyzed in eight different tissues (caruncular endometrium, intercaruncular endometrium, corpus luteum, ovary, oviduct, mammary gland, embryonic disc and trophoblast) from three pregnant dairy cows. Three programs—GeNorm, NormFinder and Bestkeeper—were used to identify the best RGs. According to all three programs, the most stable RG was CNOT11, whereas the least stable RGs were GAPDH and HPRT1. GeNorm analysis showed that a combination of five RGs (SDHA, PPIA, CNOT11, RPS9 and RPL19) was necessary for appropriate data normalization. However, NormFinder analysis indicated that the combination of CNOT11 and PPIA was the most suitable. When target genes were normalized to these RGs, the relative expression of the Radical S-adenosyl methionine domain containing 2 gene was not affected by the choice of RGs, whereas a large difference was observed in the expression profile of the Nuclear erythroid2-related factor 2 gene between the most stable RGs and least stable RGs. Conclusions: The results indicate that careful selection of RGs is crucial under different conditions, especially for target genes with relatively small fold changes. Furthermore, the results provide useful information for the selection of RGs for evaluating genes affecting bovine reproduction.

Relative Expression of Mouse Udp-glucuronosyl Transferase 2b1 Gene in the Livers, Kidneys, and Hearts: The Influence of Nonsteroidal Anti-inflammatory Drug Treatment

2019 ◽  
Vol 20 (11) ◽  
pp. 918-923 ◽  
Author(s):  
Yazun Jarrar ◽  
Qais Jarrar ◽  
Mohammad Abu-Shalhoob ◽  
Abdulqader abed ◽  
Esra'a Sha'ban
Keyword(s):  
Gene Expression ◽  
Strongly Correlated ◽  
Chain Reaction ◽  
Inflammatory Drug ◽  
Relative Expression ◽  
Elimination Half ◽  
Endogenous Compounds ◽  
Polymerase Chain ◽  
Anti Inflammatory ◽  
Glucuronosyl Transferase

Background: Mouse Udp-glucuronosyl Transferase (UGT) 2b1 is equivalent to the human UGT2B7 enzyme, which is a phase II drug-metabolising enzyme and plays a major role in the metabolism of xenobiotic and endogenous compounds. This study aimed to find the relative expression of the mouse ugt2b1 gene in the liver, kidney, and heart organs and the influence of Nonsteroidal Anti-inflammatory Drug (NSAID) administration. Methods: Thirty-five Blab/c mice were divided into 5 groups and treated with different commonly-used NSAIDs; diclofenac, ibuprofen, meloxicam, and mefenamic acid for 14 days. The livers, kidneys, and hearts were isolated, while the expression of ugt2b1 gene was analysed with a quantitative real-time polymerase chain reaction technique. Results: It was found that the ugt2b1 gene is highly expressed in the liver, and then in the heart and the kidneys. NSAIDs significantly upregulated (ANOVA, p < 0.05) the expression of ugt2b1 in the heart, while they downregulated its expression (ANOVA, p < 0.05) in the liver and kidneys. The level of NSAIDs’ effect on ugt2b1 gene expression was strongly correlated (Spearman’s Rho correlation, p < 0.05) with NSAID’s lipophilicity in the liver and its elimination half-life in the heart. Conclusion: This study concluded that the mouse ugt2b1 gene was mainly expressed in the liver, as 14-day administration of different NSAIDs caused alterations in the expression of this gene, which may influence the metabolism of xenobiotic and endogenous compounds.

scholarly journals Evolution of Antennapedia-Class Homeobox Genes

Genetics ◽  
1996 ◽  
Vol 142 (1) ◽  
pp. 295-303 ◽  
Author(s):  
Jianzhi Zhang ◽  
Masatoshi Nei
Keyword(s):  
Gene Expression ◽  
Homeobox Genes ◽  
Amino Acid Sequences ◽  
Gene Arrangement ◽  
Gene Duplications ◽  
Group I ◽  
Group 3 ◽  
Group Ii ◽  
Anterior Posterior

Antennapedia (Antp)-class homeobox genes are involved in the determination of pattern formation along the anterior-posterior axis of the animal embryo. A phylogenetic analysis of Antp-class homeodomains of the nematode, Drosophila, amphioxus, mouse, and human indicates that the 13 cognate group genes of this gene family can be divided into two major groups, i.e., groups I and II. Group I genes can further be divided into subgroups A (cognate groups 1–2), B (cognate group 3), and C (cognate groups 4–8), and group II genes can be divided into subgroups D (cognate groups 9–10) and E (cognate groups 11–13), though this classification is somewhat ambiguous. Evolutionary distances among different amino acid sequences suggest that the divergence between group I and group II genes occurred ∼1000 million years (MY) ago, and the five different subgroups were formed by ∼600 MY ago, probably before the divergence of Pseudocoelomates (e.g., nematodes) and Coelomates (e.g., insects and chordates). Our results show that the genes that are phylogenetically close are also closely located in the chromosome, suggesting that the colinearity between the gene expression and gene arrangement was generated by successive tandem gene duplications and that the gene arrangement has been maintained by some sort of selection.

Study of Dosage Compensation in Drosophila

Genetics ◽  
2003 ◽  
Vol 165 (3) ◽  
pp. 1167-1181
Author(s):  
Pei-Wen Chiang ◽  
David M Kurnit
Keyword(s):  
Gene Expression ◽  
X Chromosome ◽  
Dosage Compensation ◽  
Qpcr Assay ◽  
Male And Female ◽  
Multiple Genes ◽  
Chromosomal Changes ◽  
Regulatory Effects

Abstract Using a sensitive RT-QPCR assay, we analyzed the regulatory effects of sex and different dosage compensation mutations in Drosophila. To validate the assay, we showed that regulation for several genes indeed varied with the number of functional copies of that gene. We then confirmed that dosage compensation occurred for most genes we examined in male and female flies. Finally, we examined the effects on regulation of several genes in the MSL pathway, presumed to be involved in sex-dependent determination of regulation. Rather than seeing global alterations of either X chromosomal or autosomal genes, regulation of genes on either the X chromosome or the autosomes could be elevated, depressed, or unaltered between sexes in unpredictable ways for the various MSL mutations. Relative dosage for a given gene between the sexes could vary at different developmental times. Autosomal genes often showed deranged regulatory levels, indicating they were in pathways perturbed by X chromosomal changes. As exemplified by the BR-C locus and its dependent Sgs genes, multiple genes in a given pathway could exhibit coordinate regulatory modulation. The variegated pattern shown for expression of both X chromosomal and autosomal loci underscores the complexity of gene expression so that the phenotype of MSL mutations does not reflect only simple perturbations of genes on the X chromosome.

scholarly journals Modulation of the Tissue Expression Pattern of Zebrafish CRP-Like Molecules Suggests a Relevant Antiviral Role in Fish Skin

Biology ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 78
Author(s):  
Melissa Bello-Perez ◽  
Mikolaj Adamek ◽  
Julio Coll ◽  
Antonio Figueras ◽  
Beatriz Novoa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bacterial Infections ◽  
Quantitative Polymerase Chain Reaction ◽  
Tissue Expression ◽  
Interferon Response ◽  
Fish Skin ◽  
Tissue Expression Pattern ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Antiviral Role

Recent studies suggest that short pentraxins in fish might serve as biomarkers for not only bacterial infections, as in higher vertebrates including humans, but also for viral ones. These fish orthologs of mammalian short pentraxins are currently attracting interest because of their newly discovered antiviral activity. In the present work, the modulation of the gene expression of all zebrafish short pentraxins (CRP-like proteins, CRP1-7) was extensively analyzed by quantitative polymerase chain reaction. Initially, the tissue distribution of crp1-7 transcripts and how the transcripts varied in response to a bath infection with the spring viremia of carp virus, were determined. The expression of crp1-7 was widely distributed and generally increased after infection (mostly at 5 days post infection), except for crp1 (downregulated). Interestingly, several crp transcription levels significantly increased in skin. Further assays in mutant zebrafish of recombinant activation gene 1 (rag1) showed that all crps (except for crp2, downregulated) were already constitutively highly expressed in skin from rag1 knockouts and only increased moderately after viral infection. Similar results were obtained for most mx isoforms (a reporter gene of the interferon response), suggesting a general overcompensation of the innate immunity in the absence of the adaptive one.

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