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

A. Malgoyre (1) , Sébastien Banzet (1) , Catherine Mouret (2) , A. Xavier Bigard (1) , André Peinnequin (2)

1 Département des Facteurs Humains
2 Département de Radiobiologie et Radiopathologie

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.

Mots clés: Locked nucleic acid (LNA); Polymerase chain reaction (PCR); Interleukin-6 (IL6); Hybridization; LNA primer design; Low expressed mRNA

Domaines: Biochimie, Biologie Moléculaire

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, 2007, 354 (1), pp.246-52. ⟨10.1016/j.bbrc.2006.12.194⟩. ⟨inserm-00388011⟩