ArticleBMC research notes2013
Reverse transcription quantitative real-time polymerase chain reaction reference genes in the spared nerve injury model of neuropathic pain: validation and literature search.
Article in BMC research notes, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 20 papers.
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20 citing papers in PubMed, 35 citations in OpenAlex.
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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.Research in veterinary science · 2017Article
- Activation of KCNQ Channels Suppresses Spontaneous Activity in Dorsal Root Ganglion Neurons and Reduces Chronic Pain after Spinal Cord Injury.Journal of neurotrauma · 2017Article
- Reciprocal relationship between membrane type 1 matrix metalloproteinase and the algesic peptides of myelin basic protein contributes to chronic neuropathic pain.Brain, behavior, and immunity · 2017Article
- NR2B Expression in Rat DRG Is Differentially Regulated Following Peripheral Nerve Injuries That Lead to Transient or Sustained Stimuli-Evoked Hypersensitivity.Frontiers in molecular neuroscience · 2016Article
- Artemin promotes functional long-distance axonal regeneration to the brainstem after dorsal root crush.Proceedings of the National Academy of Sciences of the United States of America · 2015Article
- New insights on the standardization of peripheral nerve regeneration quantitative analysis.Neural regeneration research · 2015Article
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- Validation of four reference genes for quantitative mRNA expression studies in a rat model of inflammatory injury.Molecular pain · 2014Article
- Persistent pain after spinal cord injury is maintained by primary afferent activity.The Journal of neuroscience : the official journal of the Society for Neuroscience · 2014Article
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3 authors at 1 institution in 1 country.
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No grant is acknowledged in the PubMed record.
Abstract
backgroundThe reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a widely used, highly sensitive laboratory technique to rapidly and easily detect, identify and quantify gene expression. Reliable RT-qPCR data necessitates accurate normalization with validated control genes (reference genes) whose expression is constant in all studied conditions. This stability has to be demonstrated.We performed a literature search for studies using quantitative or semi-quantitative PCR in the rat spared nerve injury (SNI) model of neuropathic pain to verify whether any reference genes had previously been validated. We then analyzed the stability over time of 7 commonly used reference genes in the nervous system - specifically in the spinal cord dorsal horn and the dorsal root ganglion (DRG). These were: Actin beta (Actb), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal proteins 18S (18S), L13a (RPL13a) and L29 (RPL29), hypoxanthine phosphoribosyltransferase 1 (HPRT1) and hydroxymethylbilane synthase (HMBS). We compared the candidate genes and established a stability ranking using the geNorm algorithm. Finally, we assessed the number of reference genes necessary for accurate normalization in this neuropathic pain model.
resultsWe found GAPDH, HMBS, Actb, HPRT1 and 18S cited as reference genes in literature on studies using the SNI model. Only HPRT1 and 18S had been once previously demonstrated as stable in RT-qPCR arrays. All the genes tested in this study, using the geNorm algorithm, presented gene stability values (M-value) acceptable enough for them to qualify as potential reference genes in both DRG and spinal cord. Using the coefficient of variation, 18S failed the 50% cut-off with a value of 61% in the DRG. The two most stable genes in the dorsal horn were RPL29 and RPL13a; in the DRG they were HPRT1 and Actb. Using a 0.15 cut-off for pairwise variations we found that any pair of stable reference gene was sufficient for the normalization process.
conclusionsIn the rat SNI model, we validated and ranked Actb, RPL29, RPL13a, HMBS, GAPDH, HPRT1 and 18S as good reference genes in the spinal cord. In the DRG, 18S did not fulfill stability criteria. The combination of any two stable reference genes was sufficient to provide an accurate normalization.
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