Analysis of Housekeeping Genes for Accurate Normalization of qPCR Data During Early Postnatal Brain Development
Maturation of the neocortex during the first three postnatal weeks is a complex process that is characterized by different time courses of maturation of different areas of the neocortex. Analysis of gene expression using quantitative PCR after reverse transcription during this period of ontogeny and comparison of different cortical areas require optimal selection of reference genes for correct normalization of the data. Here, we compared expression of nine reference genes in the somatosensory and visual areas of the neocortex at the age of 5, 8, 10, 13, and 20 days. Using widely used GeNorm and NormFinder applications, as well as a novel approach, we compared stability of expression of GAPDH, YWHAZ, TFR1, RPS18, Rn18S, HPRT1, KIF5C, OSBP, and UQCRFS1. We found that, in both neocortical areas studied, YWHAZ and UQCRFS1 are the best reference genes whereas GAPDH and TFR1 are also stably expressed in the somatosensory cortex and OSBP is stable in the visual cortex. Additionally, analysis of stability of expression of these genes by a novel approach showed that the expression of these genes is stable during the entire period from the 5th to the 20th postnatal days.
KeywordsqPCR Neocortex Normalization Neurons Reference genes
This study was supported by the Russian Foundation for Basic Research, project no. 15-04-06115-a.
Compliance with Ethical Standards
All experiments were conducted in accordance with the guidelines of the animal ethics committee of the Institute of Higher Nervous Activity and Neurophysiology of the Russian Academy of Sciences following the Directive 2010/63/EU of the European Parliament and the EU Council from Sept. 22, 2010.
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