scholarly journals Real-Time PCR Data Processing Shown by the Analysis of Colorectal Specific Candidate Genes, ERCC1, RRM1 and TS in Relation to β2M as Endogenous Control

2012 ◽  
Vol 2 (1) ◽  
pp. 139-159 ◽  
Author(s):  
Melanie Demes ◽  
Holger Bartsch ◽  
Stefanie Scheil-Bertram ◽  
Ralph Mücke ◽  
Annette Fisseler-Eckhoff
Keyword(s):  
Data Processing ◽  
Real Time ◽  
Candidate Genes ◽  
Real Time Pcr ◽  
Endogenous Control

scholarly journals A new set of endogenous control genes for use in quantitative real-time PCR experiments show that formin Ldia2dex transcripts are enriched in the early embryo of the pond snail Lymnaea stagnalis (Panpulmonata)

2019 ◽  
Author(s):  
Harriet F Johnson ◽  
Angus Davison
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Lymnaea Stagnalis ◽  
Model Organism ◽  
Early Embryo ◽  
Pond Snail ◽  
Endogenous Control ◽  
Quantitative Real Time Pcr ◽  
Nonsense Mediated Decay ◽  
Left Right Asymmetry

ABSTRACT Although the pond snail Lymnaea stagnalis is an emerging model organism for molecular studies in a wide variety of fields, there are a limited number of verified endogenous control genes for use in quantitative real-time PCR. As part of a larger study on snail chirality, or left–right asymmetry, we assayed gene expression in pond snail embryos. We evaluated six candidate control genes, by comparing their expression in three tissues (ovotestis, foot and embryo) and used three software programmes (geNorm, Normfinder and Bestkeeper) to do so. The specific utility of these control genes was then tested by investigating the relative expression of six experimental transcripts, including formin Ldia2, a gene that has been associated with chiral variation in L. stagnalis. All six control genes were found to be suitable for use in the three tissues tested. Of the six experimental genes, it was found that all were relatively depleted in the early embryo compared with other tissues, except the formin Ldia2 gene. Instead, transcripts of the wild-type Ldia2dex were enriched in the embryo, whereas a nonfunctional frameshifted version, Ldia2sin, was severely depleted. These differences in Ldia2sin expression were less evident in the ovotestis and were not evident in the foot tissue, possibly because nonsense-mediated decay is obscured in actively transcribing tissues. Our work provides a set of control genes that may be useful to the wider community and illustrates how these genes may be used to assay differences in expression in a variety of tissues.

scholarly journals Comprehensive Human Adipose Tissue mRNA and MicroRNA Endogenous Control Selection for Quantitative Real-Time-PCR Normalization

Obesity ◽  
2011 ◽  
Vol 19 (4) ◽  
pp. 888-892 ◽  
Author(s):  
Matt J. Neville ◽  
Jenny M. Collins ◽  
Anna L. Gloyn ◽  
Mark I. McCarthy ◽  
Fredrik Karpe
Keyword(s):  
Adipose Tissue ◽  
Real Time ◽  
Real Time Pcr ◽  
Human Adipose Tissue ◽  
Endogenous Control ◽  
Quantitative Real Time Pcr ◽  
Control Selection ◽  
Selection For

scholarly journals Clonality Test by PCR - PARR in Real Time of Canine Lymphomas

2021 ◽  
Vol 49 ◽  
Author(s):  
Saulo Romero Felix Gonçalves ◽  
Francisco De Assis Leite Souza ◽  
Pedro Paulo Feitosa de Albuquerque ◽  
Renata Pimentel Bandeira de Melo ◽  
Andrea Alice da Fonseca Oliveira
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Melting Curve ◽  
B Cell Lymphoma ◽  
Endogenous Control ◽  
Conventional Pcr ◽  
Canine Lymphoma ◽  
Two Samples ◽  
Conserved Region

Background: Lymphoma is a neoplasm of hematopoietic origin that affects canines. The proper establishment of prognosis and rapid institution of treatment are essential for a better quality of life, and immunophenotyping is one of the tools used for this purpose. The objective of this study was to perform a clonality test for immunophenotypic characterization of canine lymphomas using the polymerase chain reaction (PCR) for antigen receptor rearrangements (PARR) technique in real-time from samples fixed in formalin and embedded in paraffin.Materials, Methods & Results: The 23 analyzed samples were fixed in formalin and embedded in paraffin canine lymphoma from the Collection Laboratory of Histopathology of the Animal Pathology Area of the Departament of Veterinary Medicine - Federal Rural University of Pernambuco UFRPE. Samples were processed, their DNA was extracted, quantified, diluted, and standardized at a concentration of 50 ng/µL. After extraction, all samples were subjected to conventional PCR for endogenous control (detection of the IgM target region), in which the extracted DNA was amplified in a final volume of 25 µL. The 128 bp amplified product was detected by 1.5% agarose gel electrophoresis. Of the 23 samples analyzed for the detection of the conserved region referring to the endogenous gene, 91.30% (21/23) amplified the conserved region Cµ by conventional PCR, and two samples 8.70% (2/23) were negative. Endogenous control positive samples were subjected to real-time PCR-PARR for detection of IgH Major (LB), IgH Minor (LB), and TCRγ (LT) target regions. All reactions were performed in duplicate to reduce the risk of false-positive or false-negative results due to technical errors. Samples previously confirmed by immunohistochemistry were used as positive controls for T cell and B cell lymphoma, and MilliQ water was used as a negative control. After amplification, the melting curve gradually increased the temperature by 1C/5 s to 95C during continuous fluorescence monitoring. Of the 21 samples analyzed, 100.00% (21/21) demonstrated clonal amplification. Of these, 57.15% (12/21) were positive for phenotype B, and 42.85% (9/21) were positive for phenotype T.Discussion: Due to the importance of researching and confirming samples from files fixed and embedded in paraffin samples in laboratories, PCR-PARR is a good tool for this purpose. In the present study, real-time PCR analysis demonstrated greater sensitivity in the characterization of the immunophenotype of lymphomas from old samples fixed in formalin and embedded in paraffin. The temperature of melting curve analysismay vary depending on the amount of DNA and its quality. In the present study, it was found that the average melting temperature in the samples varied between ± 3C when compared to that in the control sample for LB and LT, 83.5C and 80C, respectively: in the literature, there is a relative difference in this temperature, which may vary up to 4C. Real-time PCR-PARR was satisfactory in the characterization of the immunophenotype of canine lymphomas from formalin-fixed and paraffin-embedded samples; therefore, its use is recommended for both retrospective studies. The use of PCR-PARR associated with histopathological and/or cytopathological examination in cases of canine lymphomas strongly helps pathologists, provide a safe establishment of the immunophenotype, minimize errors, and optimize the diagnosis, thus directly contributing to the establishment of the prognosis.Keywords: immunophenotyping, lymphoproliferative disease, real-time PCR, TCRγ, IgH.

scholarly journals Improved triplex real‐time PCR with endogenous control for synchronous identification of DNA from chicken, duck, and goose meat

2021 ◽  
Author(s):  
Guo‐Qiang Liu ◽  
Jian‐Xing Luo ◽  
Wei‐Liang Xu ◽  
Chun‐Dong Li ◽  
Yuan‐Sheng Guo ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Endogenous Control

Quantitative cytokine gene expression in human tonsils at excision and during histoculture assessed by standardized and calibrated real-time PCR and novel data processing

2003 ◽  
Vol 283 (1-2) ◽  
pp. 27-43 ◽  
Author(s):  
Athos Bonanomi ◽  
Dejan Kojic ◽  
Bettina Giger ◽  
Zaira Rickenbach ◽  
Louis Jean-Richard-Dit-Bressel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Data Processing ◽  
Real Time ◽  
Real Time Pcr ◽  
Cytokine Gene Expression ◽  
Cytokine Gene

Evaluating Precision and Accuracy When Quantifying Different Endogenous Control Reference Genes in Maize Using Real-Time PCR

2009 ◽  
Vol 57 (7) ◽  
pp. 2903-2911 ◽  
Author(s):  
Tandace A. Scholdberg ◽  
Tim D. Norden ◽  
Daishia D. Nelson ◽  
G. Ronald Jenkins
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Endogenous Control ◽  
Precision And Accuracy

scholarly journals A set of endogenous control genes for use in quantitative real-time PCR experiments reveal that the wild-type formin Ldia2 is enriched in the early pond snail embryo

2019 ◽  
Author(s):  
Harriet F. Johnson ◽  
Angus Davison
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Lymnaea Stagnalis ◽  
Model Organism ◽  
Early Embryo ◽  
Pond Snail ◽  
Endogenous Control ◽  
Wild Type ◽  
Quantitative Real Time Pcr ◽  
Nonsense Mediated Decay

ABSTRACTAlthough the pond snail Lymnaea stagnalis is an emerging model organism for molecular studies in a wide variety of fields including development, biomineralisation and neurophysiology, there are a limited number of verified endogenous control genes for use in quantitative real-time PCR (qRT-PCR). As part of larger study on snail chirality or left-right asymmetry, we wished to assay relative gene expression in pond snail embryos, so we evaluated six new candidate control genes, by comparing their expression in three tissues (ovotestis, foot, and embryo) and across three programs (geNorm, Normfinder and Bestkeeper). The specific utility of these control genes was then tested by investigating the relative expression of six experimental transcripts, including the formin Ldia2, a gene that has been associated with chirality in L. stagnalis. All six control genes were found to be suitable for use. Of the six experimental genes that were tested, it was found that all were relatively depleted in the early embryo compared with other tissues, except the formin gene Ldia2. Instead, transcripts of the wild type Ldia2dex were enriched in the embryo, whereas a non-functional frameshifted version Ldia2sin was severely depleted. These differences in Ldia2sin expression were less evident in the ovotestis and not evident in the foot tissue, suggesting that nonsense-mediated decay may be obscured in actively transcribing tissues. This work therefore provides a set of control genes that may be useful to the wider community, and shows how they may be used to assay differences in expression in the early embryo.

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