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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(More)
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.(More)
Genetic variability in the promoter and 3' region of the SNCA gene coding alpha-synuclein modulates the risk to develop sporadic Parkinson's disease (PD). Whether this is mediated by regulating alpha-synuclein expression levels remains unknown. Therefore, we analyzed levels of alpha-synuclein in blood and human post mortem brain tissue including the(More)
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(More)
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(More)
Experiments using quantitative real-time PCR to test hypotheses are limited by technical and biological variability; we seek to minimise sources of confounding variability through optimum use of biological and technical replicates. The quality of an experiment design is commonly assessed by calculating its prospective power. Such calculations rely on(More)
We propose a computing method for the estimation of real-time PCR ampli®cation ef®ciency. It is based on a statistic delimitation of the beginning of exponentially behaving observations in real-time PCR kinetics. PCR ground ¯uorescence phase, non-exponential and plateau phase were excluded from the calculation process by separate mathematical algorithms. We(More)
BACKGROUND Quantitative PCR (qPCR) is a valuable technique for accurately and reliably profiling and quantifying gene expression. Typically, samples obtained from the organism of study have to be processed via several preparative steps before qPCR. METHOD We estimated the errors of sample withdrawal and extraction, reverse transcription (RT), and qPCR(More)
BACKGROUND The EC-funded project SPIDIA is aimed to develop evidence-based quality guidelines for the pre-analytical phase of blood samples used for DNA molecular testing. To this purpose, a survey and a pan-European External Quality Assessment (EQA) were implemented. METHODS SPIDIA facility sent to all the participants the same blood sample to be(More)
Quantitative real-time PCR (qPCR) is the method of choice for specific and sensitive quantification of nucleic acids. However, data validation is still a major issue, partially due to the complex effect of PCR inhibition on the results. If undetected PCR inhibition may severely impair the accuracy and sensitivity of results. PCR inhibition is addressed by(More)