Contribution to Improving of the Quantification of Nucleic Acids using qPCR and RT-qPCR
type: Dataset
description: QPCR is currently the gold standard for DNA quantification of DNA. It can be defined as an exponential, cyclic and targeted amplification of a known DNA sequence. The exponential character of qPCR is both the cause of the sensitivity of the method but also a potential variability between samples. This variability is offset by the cyclical nature of the method that causes a synchronization of the reaction for all samples in each cycle. The targeted amplification of the selected sequence translates the specificity of the method. Nevertheless, this last property of PCR remains the least tested. The specificity of qPCR is unquestionable when the target to detect is sufficient (> 100 copies). However, for smaller quantities or in the presence of inhibitors, the specificity decreases or disappears (limits of detection and quantification). This loss of specificity will have a significant effect upon the accuracy and reproducibility of the assay to be performed. However, if non-specific hybridizations are unavoidable in these conditions, consecutive nonspecific amplification may be limited or eliminated using more specific PCR primers or less likely to produce inter-sample differences. RT-qPCR allows RNA quantification because of an initial step of reverse transcription (conversion of an RNA into a complementary DNA). However, reverse transcription is less reproducible than PCR, generating harmful inter-sample differences both in diagnosis and in the research field. In this work, I propose innovative methods improving significantly different steps of these techniques. First, I propose an improved standardization of the reverse transcription step that can significantly reduce the variability between samples; using a constant volume of RNA extract for each sample substantially improves mRNA quantification accuracy. Second, I propose a primers modification by incorporating Locked Nucleic Acid residues (LNA); this modification increases the specificity of the primers to the lim…
identifier: NNT: 2012GRENS028
issued: 2026-05-09T11:15:59.805463
language: fr
modified: 2026-05-09T11:15:59.805468
contact point: Organization
  fn: CCSD
keyword: acides-nucleiques-bloques-lna
keyword: infoeu-reposemanticsdoctoralthesis
keyword: locked-nucleic-acid
keyword: melting-temperature
keyword: miqe-guidelines
keyword: mrna-quantification
keyword: quantification-darnm
keyword: recommandations-miqe
keyword: reverse-transcription
keyword: rt-qpcr
keyword: sdvmheplife-sciences-q-biohuman-health-and-pathology
keyword: temperature-de-fusion
keyword: theses
keyword: transcription-inverse
landing page: https://theses.hal.science/tel-00870512

Contribution to Improving of the Quantification of Nucleic Acids using qPCR and RT-qPCR
type: Distribution
format: HTML
issued: 2026-05-09T11:15:59.807746
modified: 2026-05-09T11:15:59.782052
access URL: https://theses.hal.science/tel-00870512

test_moissonnage_selune
type: Agent