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BMC Research Notes

, 3:275

Quantitative real time PCR normalization and optimization


BackgroundThe efficiency of gene therapy experiments is frequently evaluated by measuring the impact of the treatment on the expression of genes of interest by quantitative real time PCR qRT-PCR and by normalizing these values to those of housekeeping HK genes constitutively expressed throughout the experiment. The objective of this work was to study the effects of muscle gene therapy on the expression of 18 S ribosomal RNA Rn18S, a commonly used HK gene.

FindingsMouse model of motor neuron disease SOD1-G93A was injected intramuscularly with Brain-derived neurotrophic factor BDNF-TTC encoding or control naked DNA plasmids. qRT-PCR expression analysis was performed for BDNF and HK genes Rn18 S, glyceraldehyde-3-phosphate dehydrogenase Gapdh and β-actin Actb. We report that elevated BDNF expression in the injected muscle was accompanied with increased Rn18 S expression, whereas Gapdh and Actb were not affected. Increased -ribosomal output- upon BDNF stimulation was supported by increased steady-state levels of ribosomal protein mRNAs.

ConclusionsRibosomal RNA transcription may be directly stimulated by administration of trophic factors. Caution should be taken in using Rn18 S as a HK gene in experiments where muscle metabolism is likely to be altered by therapeutic intervention.

Electronic supplementary materialThe online version of this article doi:10.1186-1756-0500-3-275 contains supplementary material, which is available to authorized users.

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Autor: María Moreno-Igoa - Raquel Manzano - Sara Oliván - Ana C Calvo - Janne M Toivonen - Rosario Osta

Fuente: https://link.springer.com/

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