Quantification of low-expressed mRNA using 5' LNA-containing real-time PCR primers
- Department of Human Factors, Centre de Recherches du Service de Sante des Armees, BP87, 38702 La Tronche Cedex (France)
- Department of Radiobiology, Centre de Recherches du Service de Sante des Armees, BP87, 38702 La Tronche Cedex (France)
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.
- OSTI ID:
- 20979819
- Journal Information:
- Biochemical and Biophysical Research Communications, Vol. 354, Issue 1; Other Information: DOI: 10.1016/j.bbrc.2006.12.194; PII: S0006-291X(06)02870-1; Copyright (c) 2006 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA); ISSN 0006-291X
- Country of Publication:
- United States
- Language:
- English
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