000055410 001__ 55410
000055410 005__ 20181205220213.0
000055410 0247_ $$2doi$$a10.5075/epfl-thesis-3372
000055410 02470 $$2urn$$aurn:nbn:ch:bel-epfl-thesis3372-2
000055410 02471 $$2nebis$$a5039878
000055410 037__ $$aTHESIS
000055410 041__ $$aeng
000055410 088__ $$a3372
000055410 245__ $$aStatistical and experimental analysis of quantitative PCR
000055410 269__ $$a2005
000055410 260__ $$aLausanne$$bEPFL$$c2005
000055410 300__ $$a163
000055410 336__ $$aTheses
000055410 502__ $$aOtto Hagenbüchle, Stephan Morgenthaler, Jan Ruijter, Horst Vogel
000055410 520__ $$aQuantitative PCR (qPCR) is a powerful technique that is now commonly used in many research and clinical laboratories. Although it allows precise quantification of specific DNA sequences, it is often not used at its full potential. A number of data collection and processing strategies have been described for the implementation of quantitative PCR. However, they can be experimentally cumbersome, their relative performances have not been evaluated systematically, and they often remain poorly validated statistically and/or experimentally. Highly sophisticated mathematical models have also been proposed to decipher the PCR intimate process, but most of them were never validated. Often poor knowledge of the underlying mechanisms lead to inaccurate results and misinterpretation. Our first objective has been to measure by qPCR chromatin accessibility that was probed by a DNA adenine methylase. Results showed that 2-fold variations in relative accessibility could be assessed. However, the variability of the measures lead us to question the reproducibility of the qPCR. Placing our work in a robust statistical frame work, we proceeded with the evaluation of the parameters influencing PCR efficiency. Performance of known methods were also evaluated in terms of sensitivity, precision and robustness; and compared with various improved models, based on individual or averaged efficiency values. Our results show that when accurate quantification is required, single reaction efficiencies need to be measured and averaged for a given sample and primer. Furthermore, we show that well designed primers can hold the assumption of equal efficiency and therefore that the ∆Ct model is valid for measurement of 5-fold induction of a gene expression, at the least. Finally, we tried, as much as we could, to produce an exhaustive list of pitfalls qPCR users could stumble upon and proposed solutions. Our results allow the precise evaluation of minute amount of DNA, with a predictable and realistic number of measures.
000055410 700__ $$0(EPFLAUTH)102111$$aKarlen, Yann$$g102111
000055410 720_2 $$aMermod, Nicolas$$edir.
000055410 8564_ $$s1650459$$uhttps://infoscience.epfl.ch/record/55410/files/EPFL_TH3372.pdf$$yTexte intégral / Full text$$zTexte intégral / Full text
000055410 909CO $$ooai:infoscience.tind.io:55410$$pthesis$$pthesis-bn2018$$pDOI$$qDOI2
000055410 918__ $$aSB$$bSB-SCGC$$cISIC
000055410 920__ $$a2005-12-2$$b2005
000055410 970__ $$a3372/THESES
000055410 973__ $$aEPFL$$sPUBLISHED
000055410 980__ $$aTHESIS