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.
Highlights
Real-time quantitative PCR (RT-qPCR) is a popular laboratory method for the measurement of gene expression in a wide variety of tissues in many different species (Bustin, 2000)
The quantification of suitable reference genes for expression gene analysis following peripheral nerve injury after spinal nerve ligation has been investigated in studies on rodent dorsal root ganglia (DRG) neurons (Bangaru et al, 2012) and it is reported that reference genes exhibiting good stability included glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and mitogen activated protein kinase 6
Using three different methods of analysis we have validated a set of six candidate reference genes for gene normalisation using RT-qPCR in porcine dorsal root ganglia and spinal cord tissues
Summary
Real-time quantitative PCR (RT-qPCR) is a popular laboratory method for the measurement of gene expression in a wide variety of tissues in many different species (Bustin, 2000). A varied number of reference genes including β-actin (ACTB), β-2microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosesyl transferase 1 (HPRT1), peptidyl prolyl isomerase A (PPIA), ribosomal protein L4 (RPL4), succinate dehydrogenase complex subunit A (SDHA), TATA box binding protein (TBP) and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ) have previously been investigated for their expression stability in a wide range of porcine tissues including oocytes, blood, brain, muscle, stomach, intestine, skin, cartilage, bone marrow, pancreas, kidney, liver, lung, thymus, lymph nodes, spleen and heart (Kuijk et al, 2007; Nygard et al, 2007; Uddin et al, 2011; McCulloch et al, 2012), but as yet, reference gene stability has not been examined in porcine peripheral nerve dorsal root ganglia (DRG) or spinal cord (SC) tissues, nor at different ages or developmental stage. The quantification of suitable reference genes for expression gene analysis following peripheral nerve injury after spinal nerve ligation has been investigated in studies on rodent DRG neurons (Bangaru et al, 2012) and it is reported that reference genes exhibiting good stability included GAPDH and mitogen activated protein kinase 6
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