Quantification of low-expressed mRNA using 5' LNA-containing real-time PCR primers.
Biochem Biophys Res Commun
; 354(1): 246-52, 2007 Mar 02.
Article
en En
| MEDLINE
| ID: mdl-17217915
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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Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
ARN Mensajero
/
Oligonucleótidos Antisentido
/
Cartilla de ADN
/
Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
/
Microquímica
Tipo de estudio:
Diagnostic_studies
/
Evaluation_studies
Idioma:
En
Revista:
Biochem Biophys Res Commun
Año:
2007
Tipo del documento:
Article
País de afiliación:
Francia