This project is in the beginning stages. At this point we have established several research methods and protocols, built the supporting infrastructure in terms of equipment, space in the lab, collaborative arrangements, hired and begun to train a team of scientists and support personnel to conduct the research project. Over the course of this year we have performed over 150 sequencing runs on the Illumina HiSeq 2000 and examined peripheral inflammation, peripheral nerve damage, models of arthritis, sequencing of genetically labeled neuronal populations and completed the trigeminal transcriptome in several species. We are in the process of writing multiple reports on these data. Some of the most interesting observations relate to the expression of the exact genes that mediate the actions of known analgesic drugs such as morphine, clonidine and sodium and calcium channel blockers. We can now assign which nociceptive neuronal population is controlled by which analgesic drug and, because we are examining both model species and human post-mortem tissue, we can generalize to the human and focus analgesic research on the most relevant known and novel molecular targets. The personnel now include a Ph.D. research fellow trained in biochemistry and bioinformatics, a predoctoral fellow trained in bioinformatics and computational biology, and a post-BAC IRTA with experience in RNA extraction who also will be trained in cDNA library construction and RT-PCR analysis. The laboratory has purchased two dedicated computer workstations for high-speed sequence alignment and analysis of the transcriptome libraries and constructed a third. We upgraded our Bioanalyzer for RNA quality assessment and utilize 96 and 384 well real-time PCR devices for independent verification of mRNA alterations seen with the RNA-seq methods. For sequencing we work with investigators at the National Institutes Sequencing Center (NISC) and mainly use the Illumina High-Seq 2000 platform for deep sequencing, generally in multiplex mode to analyze 6 independent samples/lane. We determined that polyA+ selection was superior to ribosomal RNA depletion for construction of the sequencing libraries. We contracted with the University of Pennsylvania to obtain canine tissue for the RNA studies. These tissues will be obtained from controls and animals with osteosarcoma euthanized because of inadequate pain control or treated with resiniferatoxin and tissues obtained at autopsy. The data obtained will allow us to test for genes activated by nociceptive input from naturally occurring bone cancer and modulated by treatment. Accrual in the canine study is nearly complete. The data will also be used for comparison to parallel studies in mouse, rat and human (although the exact models will be different). The transcriptome project is systematically investigating the first three steps in the pain pathway beginning with injured peripheral tissue or nerve, the dorsal root ganglion and the dorsal (sensory) spinal cord. The equivalent structures for the face and head are the trigeminal ganglia and the medulla (medullary dorsal horn). We have obtained and analyzed these two tissues in collaboration with Dr. Joel Kleinman of NIMH. These same tissues will be analyzed from rat, dog and monkey. Using the Bioanalyzer, we have analyzed total RNA extracted from the human medullary dorsal horn and trigeminal ganglia. After several technical improvements, analysis yielded excellent RNA integrity numbers. We have nearly completed the initial study on inflammation-induced genes in rats. This includes a systematic examination of the time course of gene regulation in inflamed peripheral tissue, DRG and dorsal horn. Comparisons will be made to several nerve injury models since these are distinct from the inflammation and the analysis of the time course of DRG gene expression following peripheral nerve injury (axotomy) has also been partially completed. The results are being assembled for publications. We have also created a web site for data sharing and this will form the basis for a publically accessible site for other groups to access the data.