Identification and validation of reference genes for expression studies in a rat model of neuropathic pain.

Neuropathic pain is triggered by damage to or as a result of the dysfunction of the somatosensory nervous system. Gene expression profiling using DNA microarray and real-time PCR have emerged as powerful tools for the elucidation of pain-specific pathways and identification of candidate biomarkers and therapeutic targets. Proper normalization of the gene expression data with stable reference genes is a prerequisite to obtaining accurate gene expression changes. We have evaluated the stability of six candidate reference genes which include three commonly used housekeeping genes (ACTB, GAPDH and HMBS) and three ribosomal protein genes (RPL3, RPL19 and RPL29) using real-time PCR in a rat model of neuropathic pain. Unexpectedly, ACTB but not GAPDH was stably expressed. In addition, we have identified RPL29 and RPL3 as novel reference genes. Normalization of expression data using GAPDH or HMBS led to overestimation of transcriptional changes. Using RPL29/RPL3/ACTB as reference genes, a number of transcripts were found to be specifically and significantly regulated in injured dorsal root ganglia. These genes may contribute to the development of neuropathic pain pathology and may serve as candidate biomarkers for potential diagnosis.