Selection of Reliable Reference Genes for Analysis of Gene Expression in Spinal Cord during Rat Postnatal Development and after Injury.

Ján Košuth,Martina Farkašovská,Juraj Ševc,Zuzana Daxnerová,Filip Mochnacký

doi:10.3390/brainsci10010006

Ján Košuth, Martina Farkašovská + Show 3 more

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https://doi.org/10.3390/brainsci10010006

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Journal: Brain sciences	Publication Date: Dec 20, 2019
Citations: 7	License type: CC BY 4.0

Affiliation: University of Pavol Jozef Šafárik

Abstract

In order to obtain unbiased results of target gene expression, selection of the most appropriate reference gene (RG) remains a key precondition. However, an experimental study focused on the validation of stably expressed RGs in the rat spinal cord (SC) during development or after spinal cord injury (SCI) is missing. In our study, we tested the stability of the expression of nine selected RGs in rat SC tissue during normal development (postnatal days 1–43, adulthood) and after minimal (mSCI) and contusion (cSCI) spinal cord injury. The following RGs were tested: common housekeeping genes of basal cell metabolism (Gapdh, Hprt1, Mapk6) and protein translation (Rpl29, Eef1a1, Eif2b2), as well as newly designed RGs (Gpatch1, Gorasp1, Cds2) selected according to the RefGenes tool of GeneVestigator. The stability of RGs was assessed by geNorm, NormFinder, and BestKeeper. All three applets favored Gapdh and Eef1a1 as the most stable genes in SC during development. In both models of SCI, Eif2b2 displayed the highest stability of expression, followed by Gapdh and Gorasp1/Hprt1 in cSCI, and Gapdh and Eef1a1 in the mSCI experiments. To verify our results, selected RGs were employed for normalization of the expression of genes with a clear biological context in the SC—Gfap and Slc1a3/Glast during postnatal development and Aif1/Iba1 and Cd68/Ed1 after SCI.

Highlights

Myriads of processes at cellular and subcellular levels shape the developing parts of the nervous system into their final complexion
We tested the stability of the expression of nine selected housekeeping genes in the spinal cord tissue of intact animals at the age of one day (P1) to six postnatal weeks (P43) and in adulthood (P120+) in the developmental part of the study; and in adult animals (P120+) subjected to mild and severe spinal cord injury (SCI)
In the case of SCI, the stability of reference gene (RG) expression was assessed both complexly for minimal SCI (mSCI) and cSCI together, as well as independently for each individual model of injury itself

Summary

Introduction

Myriads of processes at cellular and subcellular levels shape the developing parts of the nervous system into their final complexion. One can assume that an understanding of the various pathological conditions affecting the adult nervous system is inseparably linked with an understanding of its development and maturation These developmental or pathological processes could be studied on various levels by methods of molecular biology, microscopy, electrophysiology, etc. In spite of the progress and innovations in the abovementioned methods, RT-qPCR remains the method of choice for routine quantification of gene expression levels in biomedical research [1]. This is mainly due to its accessibility; the reasonable combination of easy feasibility, high sensitivity, and resolution; and the possibility to analyze and compare the expression of a single gene/relatively small set of genes in multiple samples.

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