Neuromics' Transfection Kits-Genes Studied

Delivering siRNA, miRNA, Plasmids and Viral Vectors for Gene Expression Analysis.

I have shared the many genes researchers have studied using our Transfection Kits. These include: β-arrestin, ABCA, ASIC, β-arrestin, CAV1.2, CX3CR1, DOR, EHDAC2, LOVL4, IKBKAP, K+-ATPase, KV1.1, KV9.1 , neuroligin 2, The β3 subunit of the Na+,K+-ATPase, NTS1, NAV1.8, NTS1, NOV, Raf-1, RANK, SNSR1, hTert, NOV, Survivin, TLR4, Troy and TRPV1 and More!

We can now add GPNMB to this list: 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.

Gene Expression Analysis For Neuroscientists

In Vitro and In Vivo Studies

Neuromics has a strong line up of Transfection Tools designed specifically for Neuroscientists. Neurons, Glia and Astrocytes are notoriously hard to transfect. We are proud of our track record.

This is an excellent study for Neuroscience Researchers interested in using best methods: http://emboj.embopress.org/content/embojnl/31/15/3239/DC1/embed/inline-supplementary-material-1.pdf?download=true.  Here researchers delivered 14-3-3 siRNA sub units + i-Fect intrathecally. Here's specific knockdown results:

Figure: A. Dose-dependent inhibition of 14-3-3-zeta (z) expression with anti-14-3-3-z siRNA as measured with qRT-PCR in cultured spinal neurons (n = 3 independent experiments). B. Immunolabeling for 14-3-3-z is visualized in the dorsal horn of naive rats after intrathecal injection of mismatch RNA (a, mmRNA; 2 µg in 10µL i-Fect reagent) or anti-14-3-3-z siRNA (b, anti-14-3-3-z siRNA; 2 µg in 10µL i-Fect reagent). Bar: 50 µm C. Detection in the spinal cord (a, SC) and lumbar dorsal root ganglia (b, DRG) of intrathecally injected fluorescent siRNA. Staining is seen in the dorsal horn (open star) but not in the dorsal root ganglia (filled star). Bar: 50 µm D1. Quantification of 14-3-3-z mRNA levels with qRT-PCR in the ipsilateral lumbar (L4 and L5) dorsal spinal cord of three groups of rats (n=4 in each group): sham, neuropathic (same data as Figure 1B), and neuropathic with 3 intrathecal injections of anti-14-3-3-z siRNA. The upregulation of 14-3-3 mRNA in neuropathic conditions is abolished after anti-14-3-3-z siRNA injections. D2. Same quantitative procedure carried out in the ipsilateral lumbar (L4 and L5) dorsal root ganglia. No modification was induced by intrathecal injections of anti-14-3-3-z siRNA (n=4 in each group, same data as Figure S1B for Sham and SNL).

Our customers have successfully studied many genes with our tools. Here's a sampling: ABCA, ASIC, β-arrestin, CAV, CX3CR1, DOR, ELOVL4, IKBKAP, K+-ATPase, K_V1.1, K_V9.1 ,The β3 subunit of the Na+,K+-ATPase, NTS1, NAV_1.8, NTS1, NOV, Raf-1, RANK, SNSR1, hTert,  NOV, Survivin, TLR4, Troy and  TRPV1. Here're the related publications.

i-Fect, BDNF and Irritable Bowel Syndrome (IBS)

Gene Expression Analysis Determines BDNF's Role in IBS

In this study, Researchers used Neuromics'  i-Fect^TM Transfection Kit to deliver BDNF to determine effect on visceral hypersensitivity (VHS): J. H. Winston1 Q. Li1, S. K. Sarna1. Chronic prenatal stress epigenetically modifies spinal cord BDNF expression to induce sex-specific visceral hypersensitivity in offspring. Article first published online: 4 MAR 2014. Neurogastroenterology & Motility. DOI: 10.1111/nmo.12326. The Journal of Pain, Volume 14, Issue 11, November 2013, Pages 1485–1491 http://dx.doi.org/10.1016/j.jpain.2013.07.007

Intrathecal treatment with brain-derived neurotrophic factor (BDNF) antagonists reduced VMR to colorectal distension (CRD) in female chronic prenatal stress (CPS)+HeICS rats. (A) Graph shows that intrathecal administration of BDNF antagonist trkBFc in female CPS rats significantly decreased VMR to CRD, 24 h following adult 29 HeICS (two-way repeated measures ANOVA found a significant main effect of treatment, F1,53 = 10.4, p = 0.015; post hoc tests found significant differences at 30, 40, 50, and 60 mmHg, n = 4). (B) Graph shows that intrathecal administration of BDNF siRNA in female CPS rats significantly decreased VMR to CRD, 24 h following adult 29 HeICS (two-way repeated measures ANOVA: treatment 9 pressure interaction, F1,77 = 3.49, p = 0.008, tukey post hoc tests found significance at 30 mmHg, p = 0.013 and at 40, 50 50, 80 mmHg, p < 0.001, n = 7 Ctr., n = 6 BDNF siRNA). (C) Western blot shows a significant decrease in spinal cord BDNF protein expression in rats treated with BDNF siRNA (*p < 0.05).

Conclusion: Chronic prenatal stress followed by a second exposure to HeICS in adult offspring exacerbated visceral hypersensitivity (VHS) greater in female offspring that persisted longer than in male offspring. Chronic prenatal stress up-regulated BDNF expression in the lumbar-sacral dorsal horn that correlated with the exacerbation of VHS in female, but not in male offspring by increasing RNA Pol II binding and histone H3 acetylation, and decreasing histone deacetylase 1 association with the core promoter of BDNF in female offspring.

Using i-Fect to Knock Down GLT-1 Gene in vivo

Pain Researchers continue to use our i-Fect^TM Transfection Kit for Gene Expression Analysis

Valproate Prevents Dysregulation of Spinal Glutamate and Reduces the Development of Hypersensitivity in Rats After Peripheral Nerve Injury. The Journal of Pain, Volume 14, Issue 11, November 2013, Pages 1485–1491 http://dx.doi.org/10.1016/j.jpain.2013.07.007.

Researchers use the kit to deliver Glutamate Receptor 1 siRNA in vivo.
The present study examined whether the histone deacetylase inhibitor valproate prevents downregulation of glutamate transporters in the primary cultured astrocytes and in the spinal cord after L5-L6 spinal nerve ligation (SNL) and whether this action of valproate on spinal glutamate transporters prevents spinal glutamate dysregulation and development of hypersensitivity after SNL. In cultured astrocytes, valproate prevented downregulation of glutamate transporter-1 (GLT-1) and glutamate-aspartate transporter in a concentration-dependent manner. Repeated oral administration of valproate reduced the development of hypersensitivity and prevented the downregulation of spinal GLT-1 and glutamate-aspartate transporter expression in rats after SNL, but did not affect mechanical nociception and expression of those transporters in normal rats. Valproate's effects on hypersensitivity and spinal GLT-1 expression in SNL rats were blocked by intrathecal administration of the selective GLT-1 blocker dihydrokainic acid or the GLT-1 selective small interfering RNA (siRNA). Extracellular glutamate concentration in the spinal cord, measured by microdialysis, was increased in animals with SNL or after GLT-1 selective siRNA treatment, and valproate prevented the SNL-induced glutamate increase. These results suggest that valproate reduces the development of chronic pain after nerve injury in part by preventing downregulation of glutamate transporters, especially GLT-1, to maintain normal extracellular glutamate concentrations in the spinal cord.

i-Fect siRNA Transfection Successes

Meeting your Gene Expression Analysis Requirements.

I have posted many success stories resulting from use of  Neuromics'  Neuromics' i-Fect^TM siRNA Transfection Kit. It has been 3 months since my last posting so here I would like to post some recent highlights.

Here I feature customer publication demonstrating:

1. Down regulation of Neuroligin 2-data showed unexpected upregulation and pronociceptive effects of the “inhibitory” NL2 in neuropathic pain, suggesting a functional shift of NL2 from inhibition to excitation that changed the synaptic ratio toward higher excitation.
2. Role of IGFPB5 for survival of neurons in post-natal development.
3. HDAC role in Anxiety and Alcoholsim

Here're the publications:
Tiphaine Dolique, Alexandre Favereaux, Olivier Roca-Lapirot, Virginie Roques, Claire Léger, Marc Landy, Frédéric Nagy, Unexpected association of the “inhibitory” Neuroligin 2 with excitatory PSD95 in neuropathic pain, PAIN, Available online 25 July 2013, ISSN 0304-3959, http://dx.doi.org/10.1016/j.pain.2013.07.035.
(http://www.sciencedirect.com/science/article/pii/S030439591300403X)...The siRNAs were solubilized in 10 µl of i-Fect reagent (Neuromics, Edina, Minnesota, USA) following Neuromics instructions and published protocol, and applied intrathecally using a Hamilton syringe...

Masaki Ueno,Yuki Fujita, Tatsuhide Tanaka, Yuka Nakamura, Junichi Kikuta, Masaru Ishii & Toshihide Yamashita. Layer V cortical neurons require microglial support for survival during postnatal development. Nature Neuroscience 16, 543–551 (2013) doi:10.1038/nn.3358.
...vehicle (PBS) was delivered intraventricularly through the cisterna magna with a glass pipette while P3 mice were cold anesthetized. Igf1 siRNA (stealth siRNA, Invitrogen) or Igfbp5 siRNA with i-Fect reagent (Neuromics) was delivered intraventricularly through the cisterna magna at P3 twice with a 12-h interval...

Sachin Moonat, Amul J. Sakharkar, Huaibo Zhang, Lei Tanga, Subhash C. Pandey. Aberrant Histone Deacetylase2–Mediated Histone Modifications and Synaptic Plasticity in the Amygdala Predisposes to Anxiety and Alcoholism. Biological Psychiatry. doi.org/10.1016/j.biopsych.2013.01.012
...The siRNAs were dissolved in iFect solution (Neuromics, Edina)...

I will keep updating you as more pubs and data becomes available.

i-Fect Delivers IGF1 siRNA to the Mouse Brain

Intraventricular Injections Used to Study Neuronal Survival During Post Natal Development

Neuromics' i-Fect^TM Transfection Kit continue to be successfully used to deliver siRNA, shRNA and miRNA to cell cultures and the CNS in vivo (via intrathecal, epidural and intraventricular injection). Genes studied include: ABCA, ASIC, β-arrestin, CAV_1.2, IGF1, CX3CR1, DOR, ELOVL4, IKBKAP, K+-ATPase, K_V1.1, K_V9.1 ,The β3 subunit of the Na+,K+-ATPase, NTS1, NAV_1.8, NTS1, NOV, Raf-1, RANK,SNSR1, hTertTRPV1 NOV, Survivin, TLR4, Troy and TRPV1. Related Publications.

It is always an honor for one our products to be referenced in one of the Nature publications. Here researchers use i-Fect to study the impact of microglial derived IGF1 silencing on Neuronal Survival: Masaki Ueno,Yuki Fujita, Tatsuhide Tanaka, Yuka Nakamura, Junichi Kikuta, Masaru Ishii, Toshihide Yamashita. Layer V cortical neurons require microglial support for survival during postnatal development. Nature Neuroscience 16, 543–551 (2013) doi:10.1038/nn.3358. ...vehicle (PBS) was delivered intraventricularly through the cisterna magna with a glass pipette while P3 mice were cold anesthetized. Igf1 siRNA (stealth siRNA, Invitrogen) or Igfbp5 siRNA with i-Fect reagent (Neuromics) was delivered intraventricularly through the cisterna magna at P3 twice with a 12-h interval...

Images: (a) Igf1 expression (blue) and Iba1-positive microglia (brown) in P5 brain. Scale bar represents 400 μm. (b) Magnified view of dotted square in a. Scale bar represents 50 μm. (c,d) IGF1Ra expression (red) in CTIP2-positive (c) and SATB2-positive (d) layer V neurons at P5. Scale bar represents 50 μm. (e) IGF1 protein levels in medium from cultured cortical neurons, microglia and neurons with microglia in transwell systems detected by enzyme-linked immunosorbent assay (ELISA). **P < 0.01 (neuron, microglia + neuron, n = 5; microglia, n = 6 experiments; one-way ANOVA followed by Tukey-Kramer test). (f) The number of cleaved caspase-3–positive neurons in transwell systems treated with LY294002 or H-1356 or transfected to microglia with Igf1 siRNA. *P < 0.05, **P less than  0.01 (n = 3 experiments, one-way ANOVA followed by Tukey-Kramer test). (g) Neuronal phospho-AKT expression in cultured cortical neurons and those with microglia in transwell system. (h) TUNEL-positive cells in H-1356–treated cortex (36 h after treatment). Scale bar represents 100 μm. (i) The number of TUNEL-positive apoptotic cells in each layer in vehicle- (phosphate-buffered saline, PBS) or H-1356–treated mice (36 h after injection). *P < 0.05 (n = 4 brains, one-way ANOVA followed by Tukey-Kramer test). (j) Cleaved caspase-3–labeled cells (red) expressing CTIP2 (green, arrowheads). Scale bar represents 100 μm. (k,l) TUNEL-positive cells in the cortex treated with control or Igf1 siRNA (48 h after treatment). Scale bar represents 100 μm. (m) The number of TUNEL-positive apoptotic cells in each layer in control siRNA– and Igf1 siRNA–treated mice. **P less than 0.01 (n = 5 brains, one-way ANOVA followed by Tukey-Kramer test). Error bars represent s.e.m.

I will continue to post new developments and successes.

i-Fect™ Delivers you siRNA Payload

Delivering siRNA to Dorsal Root Ganglia to Silence K_V Receptors.

Our i-Fect transfection kits continue to be used to optimize delivery in vivo and into hard to transfect cells like primary neurons. In these 2 latest expamples, researchers use i-Fect to deliver siRNA to K_V Receptors in Rat DRGs. Knocking down these receptors enable the study of their role in pain modulation: John H. Winston, Sushil K. Sarna. Developmental Origins of Functional Dyspepsia-Like Gastric Hypersensitivity in Rats. Gastroenterology. Volume 144, Issue 3, March 2013, Pages 570–579.e3. dx.doi.org/10.1053/j.gastro.2012.11.001....intrathecal treatment, 2 μg of the appropriate siRNA was mixed (1:5 vol/vol) with i-Fect transfection reagent (Neuromics, Edina, MN); rats received 2 ug siRNA/10 uL/rat/injection...

Figures. siRNA-mediated knockdown of Kv1.1 expression in thoracic DRG significantly increased gastric sensitivity in naive adult rats. (A) Western blots showed a significant decrease in Kv1.1 protein in thoracic DRG (T8–T12) after intrathecal treatment with Kv1.1 siRNA but not with control siRNA. siRNA treatment did not alter TrpV1 expression (n = 5 rats each; *P < .01 vs control siRNA). (B) Naive rats treated with Kv1.1 siRNA showed a significant increase in VMR to gastric distention (n = 5 rats each, compared with pretreatment baseline; *P < .05). (C) Treatment with control siRNA had no significant effect on gastric hypersensitivity. (D) Patch clamp recordings from freshly dissociated gastric DRG neurons from FD-like and PND 10 saline-treated littermate controls showed a significant decrease in rheobase in FD-like rats (*P < .05), and (E) a significant increase in the number of action potentials elicited by current injection at 3× the rheobase in gastric DRG neurons from FD-like rats (*P < .05). (F) Sample voltage vs time traces showing action potentials evoked at ×1, ×2, and ×3 rheobase. The patch clamp data were obtained from 16 cells from 5 PND 10 saline control rats and 19 cells from 5 FD-like rats.
Tsantoulas C, Zhu L, Shaifta Y, Grist J, Ward JP, Raouf R, Michael GJ, McMahon SB. Sensory neuron downregulation of the Kv9.1 potassium channel subunit mediates neuropathic pain following nerve injury. J Neurosci. 2012 Nov 28;32(48):17502-13. doi: 10.1523/JNEUROSCI.3561-12.2012..."I'd just like to notify you about a recent paper from my team which utilised iFect for in vivo transfection of dorsal root ganglia." Dr. Christoforos Tsantoulas, University of Cambridge...i-Fect-siRNA-mediated knock-down of Kv9.1 in naive rats led to neuropathic pain behaviors...

These add to the many publications referencing use of i-Fect to deliver siRNA. Genes studied include:  DOR, hTERT, The β3 subunit of the Na+,K+-ATPase, rSNSR1, NTS1. NAV1.8, , RANK, Toll-Like Receptors, Kv Recptors, BDNF, Ret, TRPV1, Survivin, Flaviviruses, NOV, Troy β-arrestin, TRPV1 CAV1.2 TLR4 and ASIC.