scholarly journals Prospect and Competence of Quantitative Methods via Real-time PCR in a Comparative Manner: An Experimental Review of Current Methods

2018 ◽  
Vol 11 (1) ◽  
pp. 1-11
Author(s):  
Hossein Mahboudi ◽  
Negin Mohammadizadeh Heidari ◽  
Zahra Irani Rashidabadi ◽  
Ali Houshmand Anbarestani ◽  
Soroush Karimi ◽  
...  
Keyword(s):  
Internal Control ◽  
Copy Number ◽  
Quantitative Methods ◽  
Absolute Quantification ◽  
Relative Quantification ◽  
Absolute Quantitation ◽  
Standard Curve ◽  
Qpcr Quantification ◽  
Comparative Manner

Background: There are numerous approaches dealing with relative and absolute quantitation. The methods differ in their efficiency assumption and applicability. Objective: Current methodologies and rations used in qPCR quantification were compared in an experimental study of transgenic copy number determination of a monoclonal antibody Daclizumab. Methods: With an inter and intra-methodical view, variations in relative and absolute quantification strategies were discretely extracted and compared to one another. Results: In relative quantification, six methods were studied and the ratios were computed relative to Glucagon as internal control. For Absolute quantification, the calculations were based on standard curve. Relative quantification considers the relative changes in expression levels while Absolute quantification relates the PCR signal to input copy number with a calibration curve. Conclusion: The observed unevenness of the ratios in Relative approach pointed mainly to the efficiency changes and its calculation formula. Whereas results in Absolute approach strategies showed homogeneity which indicates the consistency of the calculation method.

scholarly journals Proteomic Analysis of Beef Tenderloin and Flank Assessed Using an Isobaric Tag for Relative and Absolute Quantitation (iTRAQ)

Animals ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 150
Author(s):  
Zhaomin Lei ◽  
Jianping Wu ◽  
Deyin Zhang ◽  
Ting Liu ◽  
Shengguo Zhao ◽  
...  
Keyword(s):  
Proteomic Analysis ◽  
Expression Patterns ◽  
Tca Cycle ◽  
Absolute Quantification ◽  
Absolute Quantitation ◽  
Oxidation Reduction ◽  
The Tca Cycle ◽  
Isobaric Tag ◽  
Isobaric Tags

Herein, we performed a proteomic analysis of tenderloin and flank steaks from Simmental cattle using the isobaric tags for a relative and absolute quantification (iTRAQ) approach. We identified 17 amino acids in both steaks, and Gly, Cys, Ile, Lys, and Pro differed most in abundance between the steak types (p < 0.05). A comparison of the expression patterns in steaks revealed 128 differentially expressed proteins (DEPs), of which 44 were up-regulated and 84 were down-regulated. Furthermore, 27 DEPs (p < 0.05) were subjected to gene ontology (GO) analysis, and many were found to be related to oxidation-reduction, metabolism, hydrogen ion transmembrane transport, transport, the tricarboxylic acid (TCA) cycle, mitochondrial electron transport, and the conversion of nicotinamide adenine dinucleotide (NADH) to ubiquinone. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis also implicated these DEPs in various signalling pathways, including oxidative phosphorylation, cardiac muscle contraction, the TCA cycle, biosynthesis, and the metabolism. These findings provide a new insight into key proteins involved in the determination of amino acid composition in beef.

Comparative Study Between Relative and Absolute BCR-ABL Transcript Quantification by Real Time PCR in Chronic Myeloid Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4248-4248
Author(s):  
Paula Oliveira Montandon Hokama ◽  
Juliana Capannacci ◽  
Newton Key Hokama ◽  
Kozue Miyashiro ◽  
Fernando Lopes Alberto ◽  
...  
Keyword(s):  
Real Time ◽  
Reference Gene ◽  
Internal Control ◽  
Real Time Pcr ◽  
Myeloid Leukemia ◽  
Target Gene ◽  
Chronic Phase ◽  
Absolute Quantification ◽  
Standard Curve ◽  
The Absolute

Abstract After the introduction of tirosine-kinase inhibitor in chronic myeloid leukemia (CML) treatment, the quantification of the level BCR/ABL positive cells has become essential. Since 1993, our lab has been using a sensitivity qualitative nested RT-PCR assay. Due to the need of tumor cells quantification in CML, we developed, in 2004, a quantitative test by real time PCR. The question was to define whether the relative or absolute quantification was the best strategy. To answer this question our lab team standardized both methods and compared them. The real time PCR (RQ-PCR) was performed in Applied Biosystems® 7500 plataform with TaqMan® probes towards b2a2, b3a2 BCR-ABL transcript and BCR reference gene, in a non multiplex assay. Two separate RQ-PCR reactions were prepared for the BCR-ABL standard and BCR standard. 129 peripheral blood samples of CML patients were tested by both relative and absolute methods. Quality control samples were analyzed in every RQ-PCR run to monitor assay performance: NTC, positive and negative control. Relative Quantification is based on the expression levels of a target gene (BCR-ABL) versus a reference gene (BCR). This method determines the mRNA level of BCR-ABL gene across mutiple samples and expresses it relative to the levels of an internal control. A pool of c-DNA of 30 patients with untreated CML in chronic phase was used as an internal control (N Engl J Med, 2003, oct 9, 349–15). This method does not require standards with known concentrations and we used Pfaffl mathematical model to calculate the expression of a target gene in relation to a reference gene (Nucl Acid Res.2001; 29:2002–7). The relative expression ratio is calculated from the real time PCR efficiencies and the crossing point of an unknown sample versus a control: Ratio= E(target) ΔCt target (controlsample)/E(reference) ΔCt reference (control-sample) Absolute Quantification determines the input copy number of the BCR-ABL transcript, usually by relating the PCR signal to a standard curve. The standard curve was constructed using plasmids. Plasmids contaning a cDNA fragment of genes under analysis (b2a2, b3a2 BCR-ABL transcript and BCR gene) were prepared by PCR cloning (Branford S, Br J Haematol, 1999; 107:508–99). A 10-fold diluition series in the range of 106 to 10 copies was prepared for the BCR-ABL transcripts and BCR gene. The results were reported as a ratio of BCR-ABL/BCR % and were expressed relative to the median of BCR-ABL transcripts in the blood of 30 patients with untreated CML in chronic phase (baseline). Results: We performmed 129 blood samples of CML patients by both the relative and the absolute RQ-PCR. The results showed a positive correlation between relative and absolute values (r = 0.969 and p =0.000). Conclusion: The results of relative and absolute RQ-PCR methods were equivalent. Although the absolute method is more frequent in literature, the relative quantification presents more simply standardization, low contamination risk since it does not work with plasmids and results as safe as the absolute RQ-PCR.

scholarly journals Determination of CYP2D6 Gene Copy Number by Pyrosequencing

2005 ◽  
Vol 51 (3) ◽  
pp. 522-531 ◽  
Author(s):  
Erik Söderbäck ◽  
Anna-Lena Zackrisson ◽  
Bertil Lindblom ◽  
Anders Alderborn
Keyword(s):  
Copy Number ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Relative Quantification ◽  
Sequence Context ◽  
Cyp2d6 Gene ◽  
Equivalent Region ◽  
Peak Pattern ◽  
Single Set

Abstract Background: Identification of CYP2D6 alleles *5 (deletion of the whole CYP2D6 gene) and *2xN (gene duplication) is very important because they are associated with decreased or increased metabolism of many drugs. The most commonly used method for analysis of these alleles is, however, considered to be laborious and unreliable. Methods: We developed a method to determine the copy number of the CYP2D6*5 and CYP2D6*2xN alleles by use of Pyrosequencing™ technology. A single set of PCR and sequencing primers was used to coamplify and sequence a region in the CYP2D6 gene and the equivalent region in the CYP2D8P pseudogene, and relative quantification between these fragments was performed. The CYP2D8P-specific Pyrosequencing peak heights were used as references for the CYP2D6-specific peak heights. Results: Analysis of 200 pregenotyped samples showed that this approach reliably resolved 0–4 genome copies of the CYP2D6 gene. In 15 of these samples, the peak pattern from one analyzed position was unexpected but could be solved by conclusive results from a second position. The method was verified on 270 other samples, of which 267 gave results that corresponded to the expected genotype. One of the samples could not be interpreted. The reproducibility of the method was high. Conclusions: CYP2D6 gene copy determination by Pyrosequencing is a reliable and rapid alternative to other methods. The use of an internal CYP2D8P control as well as generation of a sequence context ensures a robust method and hence facilitates method validation.

Rapid determination of transgene copy number in stably-transfected mammalian cells by competitive PCR

1999 ◽  
Vol 40 (3) ◽  
pp. 101-112 ◽  
Author(s):  
Ping Fu ◽  
Paul Senior ◽  
Ross T Fernley ◽  
Geoffrey W Tregear ◽  
G.Peter Aldred
Keyword(s):  
Copy Number ◽  
Mammalian Cells ◽  
Rapid Determination ◽  
Competitive Pcr ◽  
Transgene Copy Number

scholarly journals Simultaneous Determination of Nitroimidazoles and Quinolones in Honey by Modified QuEChERS and LC-MS/MS Analysis

2018 ◽  
Vol 2018 ◽  
pp. 1-12 ◽  
Author(s):  
Haiyan Lei ◽  
Jianbo Guo ◽  
Zhuo Lv ◽  
Xiaohong Zhu ◽  
Xiaofeng Xue ◽  
...  
Keyword(s):  
Recovery Rates ◽  
Inhibition Effect ◽  
Standard Curve ◽  
Modified Quechers ◽  
Relative Standard ◽  
Acacia Honey ◽  
The Matrix ◽  
Liquid Chromatography Tandem Mass ◽  
Standard Deviations

This study reports an analytical method for the determination of nitroimidazole and quinolones in honey using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A modified QuEChERS methodology was used to extract the analytes and determine veterinary drugs in honey by LC-MS/MS. The linear regression was excellent at the concentration levels of 1–100 ng/mL in the solution standard curve and the matrix standard curve. The recovery rates of nitroimidazole and quinolones were 4.4% to 59.1% and 9.8% to 46.2% with relative standard deviations (RSDs) below 5.2% and the recovery rates of nitroimidazole and quinolones by the matrix standard curve ranged from 82.0% to 117.8% and 79% to 115.9% with relative standard deviations (RSDs) lower than 6.3% in acacia and jujube honey. The acacia and jujube honeys have stronger matrix inhibition effect to nitroimidazole and quinolones residue; the matrix inhibition effect of jujube honey is stronger than acacia honey. The matrix standard curve can calibrate matrix effect effectively. In this study, the detection method of antibiotics in honey can be applied to the actual sample. The results demonstrated that the modified QuEChERS method combined with LC-MS/MS is a rapid, high, sensitive method for the analysis of nitroimidazoles and quinolones residues in honey.

scholarly journals Determination of the Absolute Number of Transgene Copies in CMVFUT Transgenic Pigs

2012 ◽  
Vol 12 (3) ◽  
pp. 349-356 ◽  
Author(s):  
Daniel Lipiński ◽  
Joanna Zeyland ◽  
Andrzej Pławski ◽  
Ryszard Słomski
Keyword(s):  
Copy Number ◽  
Transgenic Animals ◽  
Promoter Sequence ◽  
Absolute Number ◽  
Transgenic Pigs ◽  
Cmv Promoter ◽  
Genetic Construct ◽  
Coding Sequence ◽  
The Absolute

Determination of the Absolute Number of Transgene Copies in CMVFUT Transgenic PigsThe aim of this research was to determine the number of transgene copies in the DNA of transgenic pigs. The copy number of the transgene was analysed in the transgenic animals with introduced pCMVFUT genetic construct containing a coding sequence of human H transferase under a control of CMV promoter. The copy number of the transgene that had integrated with the genome of the transgenic animals was analysed by qPCR with SYBR Green dye, which enabled nonspecific double-stranded DNA detection. CMVFT-2F and CMVFT-2R primers were used to amplify a 149 bp fragment of DNA. Forward primer had a sequence complementary to a promoter sequence and reverse primer to a coding sequence of H transferase. The copy number of the transgene in the examined samples was established by plotting the CT values obtained on a standard curve, which had been set by the usage of the CT values for the successive standard dilutions with known copy number (1.438-1.431 copies). As a standard we used pCMVFut genetic construct hydrolyzed with Not I restriction enzyme to a linear form. The real-time PCR results helped to establish the range of 3 - 4 as the number of the transgene copies that had integrated to the swine genome.

Comparison and Evaluation of Organizational Transactions for Continuous Auditing and Business Compliance

2018 ◽  
Vol 9 (2) ◽  
pp. 1-23
Author(s):  
Rui Pedro Marques ◽  
Henrique Santos ◽  
Carlos Santos
Keyword(s):  
Real Time ◽  
Internal Control ◽  
Control Mechanisms ◽  
Continuous Auditing ◽  
Technical Details ◽  
Ontological Model ◽  
High Level ◽  
Execution Pattern

This article presents a comparator module which aims to compare, in real time, executions of organizational transactions with patterns of behaviors of these transaction executions, allowing the determination of which execution pattern is being followed by running each transaction. This is according to information received by the internal control mechanisms, which continuously monitors the transaction executions. A possible application using this module was deployed and results were obtained from a case study. The results prove effectiveness of the module, mainly because it is able to assess business compliance and the qualitative risk associated to each transaction execution while it is running, enabling an efficient continuous auditing application. The innovation of this article is ensured by the use of an ontological model to represent organizational transactions, which can be applicable to any type of transaction in any business area in order to audit transactions at a very low level, contrary to what happens in traditional auditing, which occurs at a high level (e.g. compare whether a completed transaction has followed a set of procedures). Besides the conceptualization, this work presents some technical details of development and discussion of results from the case study.

Quantitative Methods for the Determination of Carotenoids in the Retina

Carotenoids ◽  
2009 ◽  
pp. 75-85
Author(s):  
Richard Bone ◽  
Wolfgang Schalch ◽  
John Landrum
Keyword(s):  
Quantitative Methods
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