scholarly journals Development of a Real-Time TaqMan PCR Method for Absolute Quantification of the Biocontrol Agent Esteya vermicola

Plant Disease ◽  
2020 ◽  
Vol 104 (6) ◽  
pp. 1694-1700
Author(s):  
Hai-Hua Wang ◽  
Can Yin ◽  
Jie Gao ◽  
Ran Tao ◽  
Chun-Yan Wang ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Biocontrol Agent ◽  
Internal Standard ◽  
Absolute Quantification ◽  
Pinewood Nematode ◽  
Hyphal Length ◽  
Taqman Pcr ◽  
Esteya Vermicola ◽  
The Absolute

Esteya vermicola has been used as an effective biocontrol agent for the management of the pinewood nematode, Bursaphelenchus xylophilus. Tools for monitoring the colonization and parasitism patterns of E. vermicola are required for the development of highly effective biocontrol strategies. Because the TaqMan PCR technique is effective for quantification of species in environmental samples, a real-time PCR-based methodology was developed for absolute quantification of E. vermicola via internal standard addition and extrapolation of DNA quantity to hyphal length. Primers and a probe for the 28S ribosomal RNA gene of E. vermicola were designed, and nested TaqMan real-time PCR-based quantification was performed. In addition, internal standard-based yield measurement was correlated to the absolute quantity of target genomic DNA. Moreover, an extrapolation curve obtained by optical microscopy and image analysis of the mycelia was constructed for the measurement of fungal hyphal length. The absolute quantification method developed in the present study provides a sensitive and accurate technique to quantify fungal density in either wood or other substrate samples and can be used as an effective tool for future studies of biocontrol agents.

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.

A method for the absolute quantification of cDNA using real-time PCR

2003 ◽  
Vol 278 (1-2) ◽  
pp. 261-269 ◽  
Author(s):  
Joseph A Whelan ◽  
Nick B Russell ◽  
Michael A Whelan
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Absolute Quantification ◽  
The Absolute

scholarly journals Satellite DNA as a target for TaqMan real-time PCR detection of the pinewood nematode, Bursaphelenchus xylophilus

2007 ◽  
Vol 8 (6) ◽  
pp. 803-809 ◽  
Author(s):  
CECILE FRANÇOIS ◽  
CHANTAL CASTAGNONE ◽  
NEIL BOONHAM ◽  
JENNY TOMLINSON ◽  
REBECCA LAWSON ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Satellite Dna ◽  
Bursaphelenchus Xylophilus ◽  
Pcr Detection ◽  
Pinewood Nematode

Quantification of the biocontrol agent Trichoderma harzianum with real-time TaqMan PCR and its potential extrapolation to the hyphal biomass

2010 ◽  
Vol 101 (8) ◽  
pp. 2888-2891 ◽  
Author(s):  
Rubén López-Mondéjar ◽  
Anabel Antón ◽  
Stefan Raidl ◽  
Margarita Ros ◽  
José Antonio Pascual
Keyword(s):  
Real Time ◽  
Trichoderma Harzianum ◽  
Biocontrol Agent ◽  
Taqman Pcr

Quantitative Detection of Species-Specific DNA in Feedstuffs and Fish Meals

2005 ◽  
Vol 68 (6) ◽  
pp. 1217-1221 ◽  
Author(s):  
PAVEL KRCMAR ◽  
EVA RENCOVA
Keyword(s):  
Mitochondrial Dna ◽  
Real Time ◽  
Dna Sequences ◽  
Real Time Pcr ◽  
Single Species ◽  
Absolute Quantification ◽  
Quantitative Detection ◽  
Vertebrate Species ◽  
Target Species ◽  
Species Specific

A sensitive and rapid method for the quantitative detection of bovine-, ovine-, swine-, and chicken-specific mitochondrial DNA sequences based on real-time PCR has been developed. The specificity of the primers and probes for real-time PCR has been tested using DNA samples of other vertebrate species that may also be present in rendered products. The quantitative detection was performed with dual-labeled probes (TaqMan) using absolute quantification with external standards of single species meat-and-bone meals. This method facilitates the detection of 0.01% of the target species–derived material in concentrate feed mixtures and fish meals.

20 SIALYLTRANSFERASE GENE EXPRESSION IN GGTA1 KNOCKOUT PIGS

2016 ◽  
Vol 28 (2) ◽  
pp. 140
Author(s):  
G. A. Kim ◽  
J.-X. Jin ◽  
S. Lee ◽  
A. Oh ◽  
B. C. Lee
Keyword(s):  
Gene Expression ◽  
Somatic Cell ◽  
Real Time ◽  
Real Time Pcr ◽  
Internal Standard ◽  
Immune Rejection ◽  
Tn Antigen ◽  
Transgenic Pigs ◽  
Sialyl Tn Antigen ◽  
Sialyl Tn

It is considered that GGTA1 knockout (KO) pig production via somatic cell NT would overcome the problem of immune rejection after xenotransplantation. It is reported that although GGTA KO mice showed only a mild increase in sialyltransferase gene expression, GGTA1 deficiency in pig could increase the sialyltransferase activities, non-Gal epitope expression, consequently may raise non-Gal xenoantigenicity. Therefore, in the present study we investigated whether the expression level of Sia-containing glycoconjugate mRNA in transgenic pigs could be affected by knocking out the GGTA1 gene. Besides GGTA1 KO pigs, double genes overexpressing pigs (2TG) and GGTA1 KO with double genes overexpressing (KO+2TG) pigs were produced by somatic cell NT. For the present study, fibroblasts were isolated from wild-type pigs without gene modification, 2TG, GGTA1 KO, and KO+2TG pig. The GAPDH gene was used as an internal standard to normalise the real-time PCR (RT-qPCR) analysis reaction efficiency and to quantify mRNA in pigs-derived fibroblast. The expression levels were compared between them (RT-qPCR) in triplicate for each sample. Oligonucleotide primers for real-time PCR were designed for Hanganutziu-Deicher antigen (ST3Gal1–4, ST6Gal1) and Sialyl-Tn antigen (ST6GalNac1, ST6GalNac2, and ST6GalNac6) analysis. For statistical analysis, one-way ANOVA with Dunn’s multiple comparison test were used. The mRNA expression of GGTA1 KO and KO+2TG pig derived fibroblasts cells genes showed that ST3Gal1, ST3Gal2, ST3Gal3, and ST6Gal1 gene expression were significantly up-regulated compared to the wild and 2TG pigs (P < 0.05). However, ST3Gal4, Sialyl-Tn antigen including ST6GalNac1, ST6GalNac2, and ST6GalNac6 in KO+2TG pigs were not different compared with the wild pigs (P > 0.05), whereas only GGTA1 KO pigs showed significantly higher expressions than wild, 2TG, and KO+2TG pigs (P < 0.05). These results demonstrated that GGTA KO pig-derived cells exhibit a higher Hanganutziu-Deicher antigen on glycoprotein and glycolipid than controls, and KO+2TG pig exhibit no differences when compared with GGTA1 KO pig, indicating that they do not act as an immune antigen in xenograft. Overall, the increase in glycosyltransferase expression suggests a corresponding increase in the cell surface sialyation in GGTA KO pig cells. For xenotransplantation, KO+2TG pigs were more preferable because of absence of immune rejection for Sia-containing glycoconjugate on glycoprotein and glycolipid than GGTA KO pigs. This study was supported by the Ministry of Trade, Industry and Energy (#10048948), Korea IPET (#114059–3), Research Institute for Veterinary Science, TS Corporation, and the BK21 plus program.

scholarly journals Quantification of mRNA in Whole Blood by Assessing Recovery of RNA and Efficiency of cDNA Synthesis

2006 ◽  
Vol 52 (4) ◽  
pp. 634-642 ◽  
Author(s):  
Masato Mitsuhashi ◽  
Shigeru Tomozawa ◽  
Katsuya Endo ◽  
Atsushi Shinagawa
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Whole Blood ◽  
Ex Vivo ◽  
Statistical Significance ◽  
Absolute Quantification ◽  
External Control ◽  
Cdna Synthesis ◽  
Optimized Conditions

Abstract Background: Current gene expression analysis relies on the assumption that the isolated RNA represents all species of mRNA in proportions equal to those in the original materials. No system is available for absolute quantification of mRNA. Methods: We applied whole blood to 96-well filterplates to trap leukocytes. Lysis buffer containing cocktails of specific reverse primers and known concentrations of synthetic external control RNA (RNA34) was added to filterplates, and cell lysates were transferred to oligo(dT)-immobilized microplates for hybridization. We then synthesized the cDNA in the oligo(dT)-immobilized microplates from these primer sites and used the cDNA for real-time PCR. RNA34 acted as a universal control, and gene amplification results were converted to quantities of mRNA per microliter of whole blood after the recovery of RNA34 in each sample was determined. Results: Under fully optimized conditions, both added RNA34 and native mRNA species exhibited ∼10% recovery from whole blood to real-time PCR. When whole blood was stimulated ex vivo, changes in gene expression as low as 30%–40% were detected with statistical significance, and the experimental CVs were low (10%–20%). Conclusion: This new system to estimate mRNA copies per microliter of whole blood may allow standardization of gene-expression–based molecular diagnostics.

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