Comparison of real-time polymerase chain reaction and end-point polymerase chain reaction for the analysis of gene expression in preimplantation embryos

2006 ◽  
Vol 18 (3) ◽  
pp. 365 ◽  
Author(s):  
Árpád Baji Gál ◽  
Joseph Wallace Carnwath ◽  
Andras Dinnyes ◽  
Doris Herrmann ◽  
Heiner Niemann ◽  
...  
Keyword(s):  
Gene Expression ◽  
Polymerase Chain Reaction ◽  
Real Time ◽  
Reverse Transcription ◽  
Dynamic Range ◽  
Chain Reaction ◽  
Real Time System ◽  
The Real ◽  
End Point ◽  
Polymerase Chain

The aim of the present study was to compare real-time polymerase chain reaction (PCR) and end-point PCR with respect to their suitability for the analysis of gene expression in samples in which the number of cells is limited; for example, in studies of preimplantation embryonic development and to determine the variability of the real-time reverse transcription–PCR assay. The sensitivity, dynamic range and precision of both PCR systems were compared using a single mouse liver cDNA standard. The real-time system was 100-fold more sensitive than the end-point system and had a dynamic range of more than four orders of magnitude. The linear range for end-point PCR extended for two orders of magnitude using a fixed end-point of 31 cycles. The percentage standard error of the mean based on 30 replicates was 0.14% of the threshold cycle (Ct) value for the real-time system and 6.8% for the end-point fluorescence intensity. The coefficients of variation (CV) for reverse transcription combined with real-time analysis and the complete gene expression protocol consisting of mRNA isolation, reverse transcription and real-time PCR analysis were 0.6% and 1.4% of the Ct values, respectively. The present paper details, for the first time, measurement of the biological variation of individual mammalian oocytes. The CV was 1.8% of the Ct value for expression analysis of six bovine oocytes. The results are discussed in relation to the analysis of gene expression in preimplantation embryo development.

scholarly journals 210 COMPARISON OF REAL-TIME PCR AND END-POINT PCR FOR ANALYSIS OF GENE EXPRESSION IN PREIMPLANTATION EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 255
Author(s):  
A. Baji Gal ◽  
J.W. Carnwath ◽  
A. Dinnyes ◽  
D. Herrmann ◽  
C. Wrenzycki ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Reverse Transcription ◽  
Real Time Pcr ◽  
Dynamic Range ◽  
Point System ◽  
Time System ◽  
Real Time System ◽  
The Real ◽  
End Point

The purpose of this study was to compare real-time PCR and end-point PCR with respect to their suitability for the analysis of gene expression in samples in which the number of cells is limited, for example, in studies of pre-implantation embryonic development. The real-time instrument was a LightCycler® from Roche Diagnostics (Budaors, Hungary) which is a capillary-based PCR system. Primers for histone H2A (housekeeping gene) were used for all PCR reactions. The end-point PCR system included an MJ Research PT-100 thermocycler, agarose gel electrophoresis, ethidium bromide staining, and image acquisition with a 12-bit CCD camera and densitometry. The sensitivity, dynamic range, and standard error of both PCR systems were compared using a single stock solution of cDNA. The more precise real-time PCR system was then used to determine the precision of a protocol for reverse transcription and the precision of a complete gene expression protocol including mRNA purification and reverse transcription. The real-time system was 100 times more sensitive than the end-point system and had a dynamic range of more than four orders of magnitude. The linear range for end-point PCR was extended for two orders of magnitude using a fixed end-point of 31 cycles. The standard error of the mean based on 30 replicates was 0.14% for the real-time system and 6.8% for the end-point system. The standard deviations for reverse transcription combined with real-time analysis and for the complete gene expression protocol were 0.6% and 1.4%, respectively. The t standard deviation was 1.8% for expression analysis of 6 bovine oocytes. In conclusion, real-time PCR system has advantages in sensitivity, dynamic range, and precision of measurement. New research areas which involve subtle changes in expression reprogramming or the analysis of low copy number transcripts (even from single cells and embryos) clearly benefit from the advent of real-time PCR analysis. However, when genes with high transcription levels are analyzed, the amount of cDNA taken from the reverse transcription reaction can be adjusted to lie within the operating range of end-point PCR. Pooling embryos is a valuable approach for both methods when the goal is to determine the behavior of the average embryo rather than variation between embryos. In many cases, the magnitude of biologically significant expression changes is so great that the higher levels of precision afforded by real-time PCR are not essential for the analysis. This work was funded by the Bilateral Scientific and Technological Collaboration Agreement (TET) between Hungary and Germany (TET D-6/01) and by the National Office of Research and Technology (NKTH) (BIO-00017/2002).

scholarly journals Molecular diagnosis of Anaplasma marginale in cattle: quantitative evaluation of a real-time PCR (Polymerase Chain Reaction) based on msp5 gene

2014 ◽  
Vol 34 (1) ◽  
pp. 29-33 ◽  
Author(s):  
Gisele M. Bacanelli ◽  
Carlos A. N. Ramos ◽  
Flábio R. Araújo
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Anaplasma Marginale ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Chain Reaction ◽  
The Real ◽  
End Point ◽  
Polymerase Chain

The rickettsia Anaplasma marginale is considered the main agent of bovine anaplasmosis. Due the nonspecific clinical signs of the anaplasmosis, the diagnosis of infection depends of laboratory confirmation. In recent years, molecular diagnostic methods have been used to detect A. marginale in cattle. However, the existence of a large number of assays of different sensitivity and cost makes the choice of an appropriate test difficult. In the present study, a real-time Polymerase Chain Reaction (PCR) based on the msp5 target gene was quantitatively assessed and compared to an end point PCR. Both reactions were subjected to sensitivity and specificity evaluation using plasmid DNA and samples from cattle experimentally infected with A. marginale. A comparative field trial of the tests was carried out using samples of cattle from a stable enzootic area for A. marginale. The real-time PCR showed a higher sensitivity than the end point PCR. This reaction (i.e. real-time PCR) was able to detect one copy of the msp5 gene in 100 ηg of plasmidial DNA, and more than 80% of its results were positive among experimentally infected animals seven days after infection. In addition, based on in silico analysis, the real-time PCR evaluated in the present study appears to be useful for the detection of A. ovis.

Relative quantitation of p53 and MDM2 gene expression in leiomyosarcoma; real-time semi-quantitative reverse transcription-polymerase chain reaction

2001 ◽  
Vol 164 (2) ◽  
pp. 177-188 ◽  
Author(s):  
Kimitaka Miyajima ◽  
Sadafumi Tamiya ◽  
Yoshinao Oda ◽  
Toshisada Adachi ◽  
Tatsuo Konomoto ◽  
...  
Keyword(s):  
Gene Expression ◽  
Polymerase Chain Reaction ◽  
Real Time ◽  
Reverse Transcription ◽  
Chain Reaction ◽  
Mdm2 Gene ◽  
Relative Quantitation ◽  
Polymerase Chain

Expression Patterns of Kinin-Dependent Genes in Endometrial Cancer

2012 ◽  
Vol 22 (6) ◽  
pp. 937-944 ◽  
Author(s):  
Joanna Orchel ◽  
Lukasz Witek ◽  
Malgorzata Kimsa ◽  
Barbara Strzalka-Mrozik ◽  
Magdalena Kimsa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Polymerase Chain Reaction ◽  
Endometrial Cancer ◽  
Real Time ◽  
Reverse Transcription ◽  
Transcriptional Activity ◽  
Expression Patterns ◽  
Control Group ◽  
Chain Reaction ◽  
Polymerase Chain

ObjectiveThe present study has focused on the identification of the differences between expression patterns of kinin-dependent genes in endometrial cancer with the use of real-time quantitative reverse transcription polymerase chain reaction and oligonucleotide microarray.Materials and MethodsThe study group consisted of 50 endometrium samples collected from women with endometrial cancer. Gene expression of kinin receptors BR1 and BR2 was evaluated with real-time quantitative reverse transcription polymerase chain reaction. The analysis of the expression profile of genes related to the kinin mitogenic signal transduction pathway was performed using HG-U133A oligonucleotide microarrays.ResultsThe transcriptional activity of the B1 receptor for kinins increased in patients with grade 1 (G1) and grade 2 (G2) endometrial cancer when compared to the control group, whereas it decreased in patients with grade 3 (G3) endometrial cancer. The expression of the B2 receptor showed a growing trend reaching the peak in the G2, whereas G3 was characterized by a decrease in the gene transcriptional activity. Significant differential gene expression was recorded for GNB1, PRKAR1A, KRAS, MAP2K2, GNG5, MAPK1, ADCY9, GNG11, JUN, PRKCA, PRKACB, FOS, PLCB4, ADCY8, and GNG12.ConclusionThe expression changes in kinin-dependent genes might cause disturbance in the underlying biological processes, which could be important for the pathogenesis of endometrial cancer. This will eventually help to improve treatment strategies for patients with endometrial cancer in the future.

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