Pain Research Gene Expression Analysis

Potent and Proven Transfection Kits

Pain Researchers have successfully modulated 25+ genes involved in pain pathways using our Transfection Kits. Highlights include: DOR,The β3 subunit of  Na+,K+-ATPase, NTS1, NAV1.8, Kv 1.1, Kv 9.1, TROY, NOV, β-arrestin, TRPV1, CAV1.2, TLR4 and ASIC and more!  To learn more, check out our Transfection Kit Publications and Blog.

Figures: Intrathecal Kv9.1 siRNA treatment induces pain behaviors in naive rats. A, qRT-PCR quantification of Kv9.1 mRNA in rat PASMC cultures transfected with one of three Kv9.1 siRNA sequences or control siRNA. B, qRT-PCR showing Kv9.1 in vivo knock-down in L5 DRG, 4 d after intrathecal delivery of siRNA #1 compared with vehicle or matched scrambled control.  C, IHC for Kv9.1 in scrambled- and siRNA-treated DRG to determine protein knockdown. Graphs illustrate quantification of number of positive myelinated neurons and mean Kv9.1 signal intensity. D, Kv9.1 siRNA infusion inflicts a reduction in mechanical pain withdrawal thresholds. E, There was no change in heat pain thresholds after siRNA treatment. Vertical arrows on x-axis denote siRNA injections. doi: 10.1523/​JNEUROSCI.3561-12.2012.


We can now add the GPNMB gene to the list of those anaylyzed: Lili Hou, Yanfeng Zhang, Yong Yang, Kai Xiang, Qindong Tan, Qulian Guo. Intrathecal siRNA Against GPNMB Attenuates Nociception in a Rat Model of Neuropathic Pain. Journal of Molecular Neuroscience. July 2014...Ten micrograms of siRNA1- GPNMB dissolved in 30 μl i-Fect transfection reagent (Neuromics, Edina, MN, USA) was administered intrathecally once daily for 7 days, starting from 1 day before CCI surgery...
Abstract: Neuropathic pain is characterized by hyperalgesia, allodynia, and spontaneous pain. Recent studies have shown that glycoprotein nonmetastatic melanoma B (GPNMB) plays a pivotal role in neuronal survival and neuroprotection. However, the role of GPNMB in neuropathic pain remains unknown. The aim of the present study was to assess the role of GPNMB in neuropathic pain. In cultured spinal cord neurons, we used two small interfering RNAs (siRNAs) targeting the complementary DNA (cDNA) sequence of rat GPNMB that had potent inhibitory effects on GPNMB, and siRNA1-GPNMB was selected for further in vivo study as it had the higher inhibitory effect. After sciatic nerve injury in rats, the endogenous level of GPNMB was increased in a time-dependent manner in the spinal cord. Furthermore, the intrathecal injection of siRNA1-GPNMB inhibited the expression of GPNMB and pro-inflammatory factors (TNF-α, IL-1β, and IL-6) and alleviated mechanical allodynia and thermal hyperalgesia in the chronic constriction injury (CCI) model of rats. Taken together, our findings suggest that siRNA against GPNMB can alleviate the chronic neuropathic pain caused by CCI, and this effect may be mediated by attenuated expression of TNF-α, IL-1β, and IL-6 in the spinal cord of CCI rats. Therefore, inhibition of GPNMB may provide a novel strategy for the treatment of neuropathic pain.

If you would like to learn how you can optimize your gene expression analysis studies, do not hesitate to e-mail: pshuster@neuromics.com or direct line: 612-801-1007.

TRPV1 and Osteoarthritis Related Pain

Our TRPV Antibodies are widely used and frequently published. Many of these feature TRPVs' role in nociceptive pain. Specifically they play important roles in the detection of noxious stimuli and inflammatory hyperalgesia.

TRPV1 has been implicated in OA pain, both in animal models and by the finding that TRPV1 genetic variants are associated with the risk of symptomatic knee OA in humans: S Kelly, R J Chapman, S Woodhams, D R Sagar, J Turner, J J Burston, C Bullock, K Paton, J Huang, A Wong, D F McWilliams, B N Okine, D A Barrett, G J Hathway, D A Walsh, V Chapman. Increased function of pronociceptive TRPV1 at the level of the joint in a rat model of osteoarthritis pain. Ann Rheum Dis doi:10.1136/annrheumdis-2013-203413.
Methods: Rat spinal cord sections from MIA- and saline-treated rats (n=5/group) (see online supplemental methods) were incubated with a polyclonal guinea pig anti-TRPV1 antibody (1 : 500, Neuromics, Edina, Minnesota, USA catalogue number GP14100) and then with Alexa 568-conjugated goat anti-guinea pig secondary antibody (1:300, Molecular Probes). TRPV1 immunostaining was visualised with a Leica DMRB/DM4000 B fluorescence microscope and images were acquired using Openlab software (PerkinElmer)...

Images: Transient receptor potential vanilloid 1 (TRPV1) immunoreactivity in the spinal cord. TRPV1 immunofluorescence detected in superficial dorsal horn (10× magnification) in rat lumbar (L3–L5) spinal cord at day 28 post-intra-articular injection of saline (A) or mono-iodoacetate (MIA) (B). Minimum and maximum brightness values were altered (32.01 min and 90.14 max) using Image J so as to highlight the area of TRPV1 positive staining. (C) Quantification of TRPV1 immunofluorescence in superficial dorsal horn of spinal cord taken from rats at 14 or 28 days following intra-articular injection of MIA and at day 28 following intra-articular injection of saline. Data are expressed as mean and SEM (n=5 per group).

Clinical trials of oral TRPV1 antagonists have been limited by on-target-induced hyperthermia. Here experimental evidence for increased functional role of TRPV1 at the level of the joint in a model of OA pain and the demonstration that blockade of joint TRPV1 ablates sensory afferent sensitization and pain behaviour support future targeted site-specific investigations of the therapeutic potential of TRPV1 for OA pain associated with synovitis. This could be good news for OA sufferers.

P2X3 Receptor and Inflammatory Nociception

P2X3 Activates TRPA1, 5-HT3 and 5-HT1A Receptors

Endogenous ATP via activation of P2X3 Receptors contributes to inflammatory nociception in different models, including the formalin injected in subcutaneous tissue of the rat's hind paw. In this study, researchers evaluated whether TRPA1, 5-HT3 and 5-HT1A receptors, whose activation is essential to formalin-induced inflammatory nociception, are involved in the nociception induced by activation of P2X3 receptors on subcutaneous tissue of the rat's hind paw: Suzy Krimon, Dionéia Araldi, Filipe César do Prado, Cláudia Herrera Tambeli, Maria Cláudia G. Oliveira-Fusaro, Carlos Amílcar Parada. P2X3 receptors induced inflammatory nociception modulated by TRPA1, 5-HT3 and 5-HT1A receptors. Pharmacology Biochemistry and Behavior, Available online 8 October 2013. http://dx.doi.org/10.1016/j.pbb.2013.09.017. Our widely used and frequently published P2X3 R Antibody places a central role in measuring the expression of the protein...containing 5% non-fat dry milk at room temperature, followed by incubation with P2X3 rabbit polyclonal antibody (1:500; Neuromics) overnight at 4 °C, rinsed six times with TBST, and then incubated for 40 minutes in goat anti-rabbit IgG peroxidase...

Image: Neuromics' P2X3 R WB Example: Sequence‐specific siRNA‐mediated repression of P2X3. (A) P2X3 mRNA inhibition by 200 nM siRNA duplexes. Twenty‐four hours after transfection of CHO‐rP2X3 cells, P2X3‐specific mRNA was measured with Q‐PCR and plotted as percentage of mRNA detected in the control treated with Oligofectamine alone. Sequences and modifications are shown in Figure 1B and Table 1. (B) P2X3 protein reduction by 200 nM siRNA‐8646/8647, but not by its mismatch analogue siRNA‐MM‐7558/7559 or the unrelated siRNA‐7126/7127. Twenty‐four hours after transfection, protein was extracted and analysed by western blotting. P2X3‐specific immunodetection reveals expression levels as shown below (an average value from two experiments). Time points as indicated at the top. Molecular weights of two glycosylated forms of P2X3 are shown on the left. 

Conclusions: Nociceptive response intensity was measured by observing the rat's behavior and considering the number of times the animal reflexively raised its hind paw (flinches) in 60 min. Local subcutaneous administration of the selective TRPA1, 5-HT3 or 5-HT1A receptor antagonists HC 030031, tropisetron and WAY 100,135, respectively, prevented the nociceptive responses induced by the administration in the same site of the non-selective P2X3 receptor agonist αβmeATP. Administration of the selective P2X3 and P2X2/3 receptor antagonist A-317491 or pretreatment with oligonucleotides antisense against P2X3 receptor prevented the formalin-induced behavioral nociceptive responses during the first and second phases. Also, the co-administration of a subthreshold dose of αβmeATP with a subthreshold dose of formalin induced nociceptive behavior, which was prevented by local administration of tropisetron, HC 030031 or WAY 100, 135. These findings have demonstrated that the activation of P2X3 receptors induces inflammatory nociception modulated by TRPA1, 5-HT3 and 5-HT1A receptors. Also, they suggest that inflammatory nociception is modulated by the release of endogenous ATP and P2X3 receptor activation, which in turn, increases primary afferent nociceptor susceptibility to the action of inflammatory mediators via interaction with TRPA1, 5-HT3 and 5-HT1A receptors in the peripheral tissue.

I will continue to post pain and inflammation related studies that reference the use of our antibodies.