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.
Highlights
The reverse transcription quantitative real-time polymerase chain reaction (RT-Quantitative real-time PCR (qPCR)) is a widely used, highly sensitive laboratory technique to rapidly and detect, identify and quantify gene expression
Reference genes used in the SNI model We found 484 articles in the PubMed database using “spared nerve injury” as keywords
Twenty-six articles were retrieved with semi-quantitative PCR or RT-qPCR performed in the rat dorsal root ganglion (DRG), spinal cord or brain
Summary
The reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a widely used, highly sensitive laboratory technique to rapidly and detect, identify and quantify gene expression. A common approach used to elucidate such changes in molecular machinery is to explore modifications in the expression of relevant genes [5,6] These variations can be detected and quantified in a sensitive, specific way using a reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), assuming that an accurate normalization has been performed with reference genes that have proved stable in all biological replicates and experimental conditions [7,8,9,10]. The recently published guidelines on the “minimum information for publication of quantitative real-time PCR experiments” (MIQE) strongly recommended the validation of reference genes used in different cell types and conditions [7] These guidelines outline the importance of RT-qPCR accuracy and recommend following detailed procedures to produce the most robust and reproducible RT-qPCR data
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