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

DA Sandercock, JE Coe, P Di Giminiani, SA Edwards

Research output: Contribution to journalArticle

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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 16 weeks 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.
Original languageEnglish
Pages (from-to)493 - 501
Number of pages9
JournalResearch in Veterinary Science
Volume114
Early online date28 Sep 2017
DOIs
Publication statusFirst published - 28 Sep 2017

Fingerprint

Ubiquitin C
Spinal Ganglia
Peptide Elongation Factor 1
Spinal Cord
Glyceraldehyde-3-Phosphate Dehydrogenases
Swine
Succinate Dehydrogenase
beta 2-Microglobulin
Pain
Genes
Tail
RNA
Gene Expression
Housekeeping
Proteins
Protein C
Ganglia
Actins
Real-Time Polymerase Chain Reaction
Software

Bibliographical note

1026454

Keywords

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

Cite this

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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 16 weeks 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.",
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Determination of stable reference genes for RT-qPCR expression data in mechanistic pain studies on pig dorsal root ganglia and spinal cord. / Sandercock, DA; Coe, JE; Di Giminiani, P; Edwards, SA.

In: Research in Veterinary Science, Vol. 114, 28.09.2017, p. 493 - 501.

Research output: Contribution to journalArticle

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AU - Coe, JE

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