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Artículo

Characterization of specific cDNA background synthesis introduced by reverse transcription in RT-PCR assays

Adrover, Martín FedericoIcon ; Muñoz, Manuel JavierIcon ; Baez, Maria VeronicaIcon ; Thomas, J.; Kornblihtt, Alberto RodolfoIcon ; Epstein, Alberto Luis; Jerusalinsky, Diana AliciaIcon
Fecha de publicación: 12/2010
Editorial: Elsevier France-editions Scientifiques Medicales Elsevier
Revista: Biochimie
ISSN: 0300-9084
Idioma: Inglés
Tipo de recurso: Artículo publicado
Clasificación temática:
Bioquímica y Biología Molecular

Resumen

To block expression of NMDA receptor NR1 subunit, we injected into rat hippocampus a Herpes Simplex Virus type 1 derived vector bearing a sequence for NR1 antisense. RT-PCR assays with RNA from hippocampus of animals injected either with NR1 antisense vector, control vector or vehicle, showed an amplification signal compatible with NR1 antisense which could be attributed either to an endogenous NR1 antisense or to an artifact. RT-PCR was performed either with different primers or without primers in the RT, using RNA from different tissues. RNAse protection assay was carried out to characterize the amplified signal nature. Our results show that the template for the unexpected amplified fragment was NR1 mRNA currently expressed in nervous tissue. We considered this basal amplification of a mRNA in a RT-PCR assay as "background amplification". After background subtraction, a significant signal only remained when samples from NR1 antisense vector injected animals were used, demonstrating that this was the only source for NR1 antisense. Background amplification at RT in the absence of primers, can constitute a troubling factor in quantitative nucleic acid determination, leading to major interference, particularly when both sense and antisense sequences are present in the sample. Since RT introduced a significant background signal for every gene analyzed, we propose that RT must be always performed also without primers. Then, this signal should be identified, quantified and subtracted from the specific reaction amplification signal.
Palabras clave: Antisense Rna , Background Synthesis , Hsv-1 Vectors , Reverse Transcription , Rt-Pcr
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info:eu-repo/semantics/openAccess Excepto donde se diga explícitamente, este item se publica bajo la siguiente descripción: Atribución-NoComercial-SinDerivadas 2.5 Argentina (CC BY-NC-ND 2.5 AR)
Identificadores
URI: http://hdl.handle.net/11336/16323
URL: http://www.sciencedirect.com/science/article/pii/S0300908410002944
DOI: http://dx.doi.org/10.1016/j.biochi.2010.07.019
Colecciones
Articulos(IBCN)
Articulos de INST.DE BIOLO.CEL.Y NEURCS."PROF.E.DE ROBERTIS"
Articulos(IFIBYNE)
Articulos de INST.DE FISIOL., BIOL.MOLECULAR Y NEUROCIENCIAS
Articulos(OCA HOUSSAY)
Articulos de OFICINA DE COORDINACION ADMINISTRATIVA HOUSSAY
Citación
Adrover, Martín Federico; Muñoz, Manuel Javier; Baez, Maria Veronica; Thomas, J.; Kornblihtt, Alberto Rodolfo; et al.; Characterization of specific cDNA background synthesis introduced by reverse transcription in RT-PCR assays; Elsevier France-editions Scientifiques Medicales Elsevier; Biochimie; 92; 12; 12-2010; 1839-1846
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