KORNBLIHTT Alberto Rodolfo
Characterization of specific cDNA background synthesis introduced by reverse transcription in RT-PCR assays.
ADROVER MF; MUÑOZ MJ.; BAEZ MV; THOMAS J; KORNBLIHTT AR; EPSTEIN AL; JERUSALINSKY DA
ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER
Lugar: París; Año: 2010
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. However, 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. RNAse protection assay was carried out to characterize the amplified signal nature. In vivo expression of exogenous antisense was effectively discriminated from endogenous NR1 mRNA thanks to RT-real-time PCR determination and quantification of background signal. 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 performed also without primers. Then, this signal should be identified, quantified and subtracted from the specific reaction amplification signal. Altogether, our results show that the template for the unexpected amplified fragment was NR1 mRNA currently expressed in nervous tissue, and strongly suggest that mRNA was the template in every case.