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Res Vet Sci. 2017 Oct;114:493-501. doi: 10.1016/j.rvsc.2017.09.025. Epub 2017 Sep 28.

Determination of stable reference genes for RT-qPCR expression data in mechanistic pain studies on pig dorsal root ganglia and spinal cord.

Author information

1
Animal and Veterinary Science Research Group, Scotland's Rural College (SRUC), West Mains Road, Edinburgh EH16 4SA, UK. Electronic address: dale.sandercock@sruc.ac.uk.
2
Animal and Veterinary Science Research Group, Scotland's Rural College (SRUC), West Mains Road, Edinburgh EH16 4SA, UK.
3
School of Agriculture, Food and Rural Development, Newcastle University, Newcastle upon Tyne NE1 7RU, UK.

Abstract

RNA expression levels for genes of interest must be normalised with appropriate reference or "housekeeping" genes that are stably expressed across samples and treatments. This study determined the most stable reference genes from a panel of 6 porcine candidate genes: beta actin (ACTB), beta-2-microglobulin (B2M), eukaryotic elongation factor 1 gamma-like protein (eEF-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), succinate dehydrogenase complex subunit A (SDHA), Ubiquitin C (UBC) in sacral dorsal root ganglia and spinal cord samples collected from 16 tail docked pigs (2/3rds of tail amputated) 1, 4, 8 and 16weeks after tail injury (4 pigs/time point). Total RNA from pooled samples was measured by SYBRgreen real-time quantitative PCR. Cycle threshold values were analysed using geNorm, BestKeeper and NormFinder PCR analysis software. Average expression stability and pairwise variation values were calculated for each candidate reference gene. GeNorm analysis identified the most stable genes for normalisation of gene expression data to be GAPDH>eEF-1>UBC>B2M>ACTB>SDHA for dorsal root ganglia and ACTB>SDHA>UBC>B2M>GAPDH>eEF-1 for spinal cord samples. Expression stability estimates were verified by BestKeeper and NormFinder analysis. Expression stability varied between genes within and between tissues. Validation of most stably expressed reference genes was performed by normalisation of calcitonin gene related polypeptide beta (CALCB). The results show similar patterns of CALCB expression when the best reference genes selected by all three programs were used. GAPDH, eEF-1 and UBC are suitable reference genes for porcine dorsal root ganglia samples, whereas ACTB, SDHA and UBC are more appropriate for spinal cord samples.

KEYWORDS:

Dorsal root ganglia; Normalisation; Pig; RT-qPCR; Reference genes; Spinal cord

PMID:
28987956
PMCID:
PMC5667896
DOI:
10.1016/j.rvsc.2017.09.025
[Indexed for MEDLINE]
Free PMC Article

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