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Quantification of low-expressed mRNA using 5' LNA-containing real-time PCR primers.

Abstract : Real-time RT-PCR is the most sensitive and accurate method for mRNA quantification. Using specific recombinant DNA as a template, real-time PCR allows accurate quantification within a 7-log range and increased sensitivity below 10 copies. However, when using RT-PCR to quantify mRNA in biological samples, a stochastic off-targeted amplification can occur. Classical adjustments of assay parameters have minimal effects on such amplification. This undesirable amplification appears mostly to be dependent on specific to non-specific target ratio rather than on the absolute quantity of the specific target. This drawback, which decreases assay reliability, mostly appears when quantifying low-expressed transcript in a whole organ. An original primer design using properties of LNA allows to block off-target amplification. 5'-LNA substitution strengthens 5'-hybridization. Consequently on-target hybridization is stabilized and the probability for the off-target to lead to amplification is decreased.
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Contributor : Valérie Sinniger <>
Submitted on : Wednesday, September 9, 2009 - 6:09:01 PM
Last modification on : Saturday, March 6, 2021 - 10:06:02 AM
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A. Malgoyre, Sébastien Banzet, Catherine Mouret, A. Xavier Bigard, André Peinnequin. Quantification of low-expressed mRNA using 5' LNA-containing real-time PCR primers.. Biochemical and Biophysical Research Communications, Elsevier, 2007, 354 (1), pp.246-52. ⟨10.1016/j.bbrc.2006.12.194⟩. ⟨inserm-00388011⟩



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