Biomedical subjects

Ales Tichopad

Publications and source records attributed to Ales Tichopad.

5 recordsLinked to original sources

PubMed2005

Model of inhibition of Thermus aquaticus polymerase and Moloney murine leukemia virus reverse transcriptase by tea polyphenols (+)-catechin and (-)-epigallocatechin-3-gallate.

Non-nutritional polyphenolic compounds such as (+)-catechin and (-)-epigallocatechin-3-gallate (EGCG) are known as anticancer chemopreventive agents and have been utilised for medical purposes in form of tea drinking. Documented anticancer properties of these compounds result from their antioxidant effects. However, also direct alteration of an enzyme performance has been reported and deserves more attention. In this paper, a direct effect of catechin and EGCG on the performance of reverse transcription (RT) and/or polymerase chain reaction (PCR) was studied. Both tea polyphenolic compounds were added into real-time RT-PCR reactions and the fluorescence data obtained were fitted with a mathematical model. Several parameters of PCR performance were compared, obtained from the mathematical model for reactions with and without addition of (+)-catechin and EGCG. Addition of EGCG to enzyme reaction seems to inhibit the RT reaction (p < 0.05) and to slow down the DNA polymerase reaction (p < 0.001). Similarly, (+)-catechin inhibited the DNA amplification (p < 0.01) but had no effect on the RT reaction. The effects could be observed in physiological flavanol concentrations ranging from 10(-5) to 10(-8)M.

Animals↗

PubMed2004

Inhibition of real-time RT-PCR quantification due to tissue-specific contaminants.

Real-time reverse transcription-polymerase chain reaction (RT-PCR) is currently considered the most sensitive method to study low abundance gene expression. Since comparison of gene expression levels in various tissues is often the purpose of an experiment, we studied a tissue-linked effect on nucleic acid amplification. Based on the raw data generated by a LightCycler instrument, we propose a descriptive mathematical model of PCR amplification. This model allowed us to study amplification kinetics of four common housekeeping genes in total RNA samples derived from various bovine tissues. We observed that unknown tissue-specific factors can influence amplification kinetics but this affect can be ameliorated, in part, by appropriate primer selection.

Animals↗

PubMed2004

Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper--Excel-based tool using pair-wise correlations.

The stability of standard gene expression is an elementary prerequisite for internal standardisation of target gene expression data and many so called housekeeping genes with assumed stable expression can exhibit either up- or down-regulation under some experimental conditions. The developed, and herein presented, software called BestKeeper determines the best suited standards, out of ten candidates, and combines them into an index. The index can be compared with further ten target genes to decide, whether they are differentially expressed under an applied treatment. All data processing is based on crossing points. The BestKeeper software tool was validated on four housekeeping genes and 10 members of the somatotropic axis differentially expressed in bovine corpora lutea total RNA. The BestKeeper application and necessary information about data processing and handling can be downloaded on http://www.wzw.tum.de/gene-quantification/bestkeeper.html.

Animals↗

PubMed2003

Standardized determination of real-time PCR efficiency from a single reaction set-up.

We propose a computing method for the estimation of real-time PCR amplification efficiency. It is based on a statistic delimitation of the beginning of exponentially behaving observations in real-time PCR kinetics. PCR ground fluorescence phase, non-exponential and plateau phase were excluded from the calculation process by separate mathematical algorithms. We validated the method on experimental data on multiple targets obtained on the LightCycler platform. The developed method yields results of higher accuracy than the currently used method of serial dilutions for amplification efficiency estimation. The single reaction set-up estimation is sensitive to differences in starting concentrations of the target sequence in samples. Furthermore, it resists the subjective influence of researchers, and the estimation can therefore be fully instrumentalized.

Algorithms↗

PubMed2003

Tissue-specific expression pattern of bovine prion gene: quantification using real-time RT-PCR.

In recent studies PrP mRNA was determined mostly by in situ hybridisation or Northern Blot analysis--methods not suitable for absolute quantification of mRNA copy numbers. Herein we report on bovine prion mRNA quantification using calibrated highly sensitive externally standardized real-time RT-PCR with LightCycler instrument. Total RNA was isolated from nine different regions of the CNS and seven peripheral organs. PrP(c) mRNA copy numbers could be determined in all tissues under study. In approval with prior studies high mRNA level was found in Neocortex and Cerebellum. Lymphatic organs showed at least as high expression levels of prion mRNA as overall brain. Lowest expression was detected in kidney. Results of our study provide insight into the involvement of different organs in pathogenesis with respect to prion mRNA expression. LightCycler technology is currently considered the most precise method for nucleic acid quantification and showed to be powerful tool for further studies on prion diseases pathogenesis.

Animals↗