Quantification of low-expressed mRNA using 5' LNA-containing real-time PCR primers.
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.
Malgoyre
A
A
Department of Human Factors, Centre de Recherches du Service de Santé des Armées, BP87, 38702 La Tronche Cédex, France.
Banzet
S
S
Mouret
C
C
Bigard
A X
AX
Peinnequin
A
A
D017931:DNA Primers / Q000235:genetics*; D008832:Microchemistry / Q000379:methods*; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D017931:DNA Primers; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; C477371:locked nucleic acid