tag:blogger.com,1999:blog-45949820803754260812024-02-19T18:50:30.873-08:00siRNA, DsiRNA and Plasmid Transfection EfficiencyBrought to you by Neuromics. This site is dedicated to improving the delivery of siRNA/DsiRNA/Plasmids in vitro & in-vivo.Neuromicshttp://www.blogger.com/profile/08144200376477478451noreply@blogger.comBlogger83125tag:blogger.com,1999:blog-4594982080375426081.post-1290104180468154152019-09-16T07:46:00.000-07:002019-09-16T08:34:51.200-07:00High Fat and Diet Induced Obesity<div dir="ltr" style="text-align: left;" trbidi="on">
<b><i>i-Fect<sup>TM</sup> Delivers Again!</i></b><br />
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Research shows that rats and humans on a high-fat diet (HFD) are less sensitive to satiety signals known to act via vagal afferent pathways. Impaired vagal afferent responsiveness to both gastric satiety hormones (CCK and leptin) and mechanical stimulation raises the possibility that changes in electrophysiological properties may be the underlying mechanism responsible for impaired vagal responsiveness to a wide variety of satiety signals.<br />
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Potassium channels play a central role. To demonstrate this researchers used our <a href="https://www.neuromics.com/NI35150" target="_blank">i-Fect siRNA Transfection Kit</a> to silence TRESK and TASK1 to understand their impact on HFD and vagal responsiveness. Gintautas Grabauskas, Xiaoyin Wu, ShiYi Zhou, JiYao Li, Jun Gao, and Chung Owyang. (2019). <a href="https://insight.jci.org/articles/view/130402" target="_blank">High-fat diet–induced vagal afferent dysfunction via upregulation of 2-pore domain potassium TRESK channel</a>. JCI Insight. https://doi.org/10.1172/jci.insight.130402.<br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjGD3aJem21cUkON0FQe0oCe8m9IuxI4SkrDOeKzyPTAHKDS0a6CxsRtR3aDR176FhHhapp8VqxdwNQ3K3AiByvWuIeimjDW1Xm6i7viijil1JzFhibun2_-0hOZb51xhxwdC77HKXoza8/s1600/TRESK+AP.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="549" data-original-width="700" height="312" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjGD3aJem21cUkON0FQe0oCe8m9IuxI4SkrDOeKzyPTAHKDS0a6CxsRtR3aDR176FhHhapp8VqxdwNQ3K3AiByvWuIeimjDW1Xm6i7viijil1JzFhibun2_-0hOZb51xhxwdC77HKXoza8/s400/TRESK+AP.jpg" width="400" /></a></div>
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<span style="font-size: x-small;">Images: (A) Representative recordings of NG neuron responses to intra–superior pancreaticoduodenal artery infusions of CCK-8 (60 pmol/kg) and leptin (60 pmol/kg) in LFD-fed or HFD-fed rats and transfected with control siRNA or TRESK siRNA. Note that CCK-8 generated significantly fewer action potentials in HFD-fed rats compared with those fed an LFD. (B) Summary histograms showing single-unit discharges in response to CCK-8 in rats given an LFD and transfected with control siRNA (n = 11) or TRESK siRNA (n = 6), HFD + control siRNA (n = 12), and HFD treated with TRESK siRNA (n = 10). Data are represented as mean ± SEM. One-way ANOVA with Bonferroni’s test, *P < 0.05 vs. LFD + control siRNA; #P < 0.05 vs. HFD + control siRNA. (C) Summary histogram showing single-unit discharges in response to leptin in rats given an LFD and transfected with control siRNA (n = 11) and TRESK siRNA (n = 5), HFD (n = 12), and HFD treated with TRESK siRNA (n = 10). Data are represented as mean ± SEM. One-way ANOVA with Bonferroni’s test, *P less than 0.05 vs. LFD + control siRNA; #P less than 0.05 vs. HFD + control siRNA. (D) Summary histogram showing CCK-AR and ObR expression in vagal sensory ganglia from LFD- and HFD-fed rats were not significantly different. HPRT was used as a loading control. Data are represented as mean ± SEM. CCK-8, cholecystokinin-8.
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Following 2 weeks of high-fat feeding, there was a significant upregulation of TRESK and a modest increase in TASK1 channels in the NG. Silencing studies indicate that the upregulation by TRESK channels is mainly responsible for a global decrease in excitability of vagal sensory neurons, which in turn dampens the response to satiety signals, such as CCK and leptin. </div>
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This make TRESK a potential therapeutic target for treating Obesity.</div>
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Neuromicshttp://www.blogger.com/profile/08144200376477478451noreply@blogger.com0tag:blogger.com,1999:blog-4594982080375426081.post-56096276664416735892019-05-09T11:19:00.000-07:002019-05-09T11:19:58.672-07:00i-Fect in Action<b><i>Knock-down of HIF-1a Attenuates Chemo Induced Pain</i></b><br />
<a href="https://journals.sagepub.com/doi/pdf/10.1177/1744806919850043" target="_blank">i-Fect <sup>TM</sup></a> is one of our original products. It has enjoyed 14 years of upping transfection percentages both <i>in-vitro</i> and<i> in-vivo</i>.<br />
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Here is a new study showing successful use of i-Fect to knock down HIF-1a <i>in-vivo</i>-Taylor Ludman and Ohannes K. Melemedjian. (2019). <a href="https://journals.sagepub.com/doi/pdf/10.1177/1744806919850043" target="_blank">Bortezomib-induced aerobic glycolysis contributes to chemotherapy-induced painful peripheral neuropathy</a>. Molecular Pain. https://doi.org/10.1177/1744806919837429.<br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgUGQZMk4ivDPjF3kb95SBxhXzrYKw9AcuHSqvrfUiDqH7MgJ1j9UY3QbiIXm51z3w7hw2xQLBnJ7m0KYNIMGHbTDfobfpy6Aq8CDDeO3yPr8o6mtTXVd97dYzHL1q4S5wjcXutLVM17Fs/s1600/i-Fect_HIF-1a.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="843" data-original-width="718" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgUGQZMk4ivDPjF3kb95SBxhXzrYKw9AcuHSqvrfUiDqH7MgJ1j9UY3QbiIXm51z3w7hw2xQLBnJ7m0KYNIMGHbTDfobfpy6Aq8CDDeO3yPr8o6mtTXVd97dYzHL1q4S5wjcXutLVM17Fs/s400/i-Fect_HIF-1a.png" width="340" /></a></div>
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<span style="font-size: x-small;">Figure 1. (a) Treatment of mice with bortezomib (Bor) for five days augmented HIF1A expression in L4-6 DRGs (*P ¼ 0.0412, five mice/
group) relative to the vehicle-treated group. (b) A schematic depicting the site of the intrathecal (IT) siRNA injection. The siRNA was
administered between the L4 and L5 vertebrae which is around 17 mm rostral to the spinal cord (SC) section innervated by the L4-6
DRGs. (c) IT injection of siRNA (1 mg in 5 ml) that targets HIF1A (siRNA) but not control siRNA (Cont), for two consecutive days,
significantly reduced the levels of HIF1A in L4-6 DRGs. (***P=0.0006, five mice/group). (d) IT siRNA did not affect HIF1A levels in L4-6 spinal cord (five mice/group). (e) After determining baseline withdrawal thresholds using von Frey filaments, male ICR mice received IP injection of vehicle or bortezomib (black arrows) and IT siRNA (blue arrows). The withdrawal thresholds were measured on days 7 to 14. IT HIF1A siRNA prevented the development of bortezomib-induced neuropathic pain. (****P less than 0.0001, five mice/group). DRG: dorsal rootganglia; HIF1A: hypoxia-inducible factor 1 alpha; IT: intrathecal; IP: intraperitoneal; siRNA: small interfering RNA.</span></div>
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This study is the first to demonstrate
that the stabilization of HIF1A expression underpins the
development of bortezomib-induced neuropathic pain.
Crucially, these findings reveal that the initiation and
maintenance of bortezomib-induced neuropathic pain
are regulated by distinct mechanisms.
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Looking to up your odds for high percentage siRNA Transfection? Try i-Fect.Neuromicshttp://www.blogger.com/profile/08144200376477478451noreply@blogger.com0tag:blogger.com,1999:blog-4594982080375426081.post-24497339683606613872019-05-09T11:02:00.000-07:002019-05-09T11:02:34.818-07:00i-Fect used to Study Angiogenesis in Brain Injury<b><i>Silencing Lactate Dehydrogenase A in vivo</i></b><br />
<b><i><br /></i></b> Pathologic CNS is characterized by neuronal damage that leads to the release of intracellular components. However, the effect of damaged cells on angiogenesis has not been clarified. This study revealed that LDHA, which is a known damage marker, promotes CNS-specific angiogenesis. LDHA-mediated angiogenesis depends on vimentin on the surface of vascular endothelial cells. The work described here proposes a novel mechanism by which neurodegeneration drives angiogenesis in the CNS.<br />
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A mixture of our <a href="https://www.neuromics.com/transfection-kits" target="_blank">i-Fect<sup>TM</sup><sup> </sup></a>and LDHA siRNA, in this study, were directly injected into mice cortexes: Hsiaoyun Lin, Rieko Muramatsu, Noriko Maedera, Hiroto Tsunematsu, Machika Hamaguchi, Yoshihisa Koyama, Mariko Kuroda, Kenji Ono, Makoto Sawada, Toshihide Yamashita. <a href="https://www.sciencedirect.com/science/article/pii/S2352396417304279" target="_blank">Extracellular Lactate Dehydrogenase A Release From Damaged Neurons Drives Central Nervous System Angiogenesis</a>. doi.org/10.1016/j.ebiom.2017.10.033.<br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjPv6trv0uSe7IWQi0Xji05UXDYazJGMNGDO5-XGs-dOpVVMsKNtrUjL-4SpNLgpS44q6oKUxYj26y_Djwm-xYT-Xk1XBIsR5TTRhnCO4emi34_eE2jUg5lJzZNn9k1qS1hO90EEiFPUCs/s1600/i-Fect-LDHA1.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="528" data-original-width="534" height="395" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjPv6trv0uSe7IWQi0Xji05UXDYazJGMNGDO5-XGs-dOpVVMsKNtrUjL-4SpNLgpS44q6oKUxYj26y_Djwm-xYT-Xk1XBIsR5TTRhnCO4emi34_eE2jUg5lJzZNn9k1qS1hO90EEiFPUCs/s400/i-Fect-LDHA1.jpg" width="400" /></a></div>
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<span style="font-size: x-small;"><i>Images: LDHA is sufficient to evoke CNS angiogenesis. (a) Representative images of CD105-labeled spinal cord sections obtained 7 days after LDHA administration. (b) Length of CD105+ neovessels around the LDHA administration site as indicated in a, n = 5 each. (c) Representative image of a Nissl-stained brain section after controlled cortical impact (CCI). (d) Representative image of the CD105-immunolabelled cerebral cortex obtained 7 days after CCI. (e) Length of CD105+ neovessels around CCI lesions as indicated in d; n = 5 each, all error bars represent the s.e.m. **P < 0.01, Student's t-tests. Scale bars, 200 μm.</i></span></div>
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The findings reveal unexpected neurovascular interactions in the injured adult CNS that may be relevant to our understanding of neuronal damage, which is a hallmark of many CNS disorders.Neuromicshttp://www.blogger.com/profile/08144200376477478451noreply@blogger.com0tag:blogger.com,1999:blog-4594982080375426081.post-58894051961026784492018-03-28T10:11:00.000-07:002018-03-28T10:11:54.470-07:00i-Fect Delivers Again!<b><i>Knocks Down Suspected Stress/Anxiety Receptor</i></b><br />
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The molecular pathogenesis underlying anxiety disorders is still unclear. Here, the authors demonstrate that myristoylated alanine-rich C-kinase substrate like 1 (MARCKSL1) overexpression in mice increases spine formation in the amygdala and induces stress hormone upregulation and anxiety-like behaviors. Suppression of MARCKSL1 in the amygdala ameliorates both the increase in stress hormones and the elevated anxiety-like behaviors. Our results indicate that MARCKSL1 expression in the amygdala plays an important role in anxiety-like behaviors.<br />
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This was proved, in part, by the knockdown of MARCKSL1 in vivo in mice using our <a href="https://www.neuromics.com/transfection-kits" target="_blank">i-Fect<sup>TM</sup></a>. Tanaka, Takashi; Shimizu, Shoko; Ueno, Masaki; Fujihara, Yoshitaka; Ikawa, Masahito; Miyata, Shingo. <a href="https://www.sciencedirect.com/science/article/pii/S2352396418301026#ab0010" target="_blank">MARCKSL1 Regulates Spine Formation in the Amygdala and Controls the Hypothalamic-Pituitary-Adrenal Axis and Anxiety-Like Behaviors</a>. https://doi.org/10.1016/j.ebiom.2018.03.018<br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhgQaHS_26RYSOmONsZAH9j79UgSUFPj9Rqejmiue0qp2vBO8G97QPG7t7y5_xcROCI7wcI0rCBDzKLhyAxV_cW53WQp-xT1JAIe51twWSEOmsfji8yztpiBS2Yxu6mQFdo5d2JKL-m1zw/s1600/i-Fect_Stress.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="701" data-original-width="803" height="348" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhgQaHS_26RYSOmONsZAH9j79UgSUFPj9Rqejmiue0qp2vBO8G97QPG7t7y5_xcROCI7wcI0rCBDzKLhyAxV_cW53WQp-xT1JAIe51twWSEOmsfji8yztpiBS2Yxu6mQFdo5d2JKL-m1zw/s400/i-Fect_Stress.jpg" width="400" /></a></div>
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<span style="font-size: x-small;"><i>Figure: Knockdown of MARCKSL1 ameliorates anxiety-like behavior in MARCKSL1 Tg mice. (A and B) For the in vivo experiment, siRNA (blue) was injected into the CeA (total 4 sites) with i-Fect siRNA transfection reagents 5 days prior to behavioral tests. (C) In situ hybridization for Marcksl1 mRNA (blue) in the amygdala after injection of Marcksl1 siRNA into the CeA of Tg/Tg mice. Scale bar, 200 μm. (D and E) Light/dark transition test and elevated plus maze performance in </i></span></div>
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<span style="font-size: x-small;"><i>MARCKSL1 knockdown mice (WT + control siRNA, n = 7; Tg/Tg + control siRNA, n = 7; Tg/Tg + Marcksl1 siRNA, n = 8).</i></span></div>
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We will continue to post new i-Fect results here.</div>
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Neuromicshttp://www.blogger.com/profile/08144200376477478451noreply@blogger.com0tag:blogger.com,1999:blog-4594982080375426081.post-84426094034551110662018-02-19T04:51:00.003-08:002018-02-19T04:53:31.582-08:00i-Fect Delivers siRNA to Study Angiogenesis in Brain Injury<b><i>Silencing Lactate Dehydrogenase A in vivo</i></b><br />
<b><i><br /></i></b> Pathologic CNS is characterized by neuronal damage that leads to the release of intracellular components. However, the effect of damaged cells on angiogenesis has not been clarified. This study revealed that LDHA, which is a known damage marker, promotes CNS-specific angiogenesis. LDHA-mediated angiogenesis depends on vimentin on the surface of vascular endothelial cells. The work described here proposes a novel mechanism by which neurodegeneration drives angiogenesis in the CNS.<br />
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A mixture of our <a href="https://www.neuromics.com/transfection-kits" target="_blank">i-Fect<sup>TM</sup><sup> </sup></a>and LDHA siRNA, in this study, were directly injected into mice cortexes: Hsiaoyun Lin, Rieko Muramatsu, Noriko Maedera, Hiroto Tsunematsu, Machika Hamaguchi, Yoshihisa Koyama, Mariko Kuroda, Kenji Ono, Makoto Sawada, Toshihide Yamashita. <a href="https://www.sciencedirect.com/science/article/pii/S2352396417304279" target="_blank">Extracellular Lactate Dehydrogenase A Release From Damaged Neurons Drives Central Nervous System Angiogenesis</a>. doi.org/10.1016/j.ebiom.2017.10.033.<br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjPv6trv0uSe7IWQi0Xji05UXDYazJGMNGDO5-XGs-dOpVVMsKNtrUjL-4SpNLgpS44q6oKUxYj26y_Djwm-xYT-Xk1XBIsR5TTRhnCO4emi34_eE2jUg5lJzZNn9k1qS1hO90EEiFPUCs/s1600/i-Fect-LDHA1.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="528" data-original-width="534" height="395" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjPv6trv0uSe7IWQi0Xji05UXDYazJGMNGDO5-XGs-dOpVVMsKNtrUjL-4SpNLgpS44q6oKUxYj26y_Djwm-xYT-Xk1XBIsR5TTRhnCO4emi34_eE2jUg5lJzZNn9k1qS1hO90EEiFPUCs/s400/i-Fect-LDHA1.jpg" width="400" /></a></div>
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<span style="font-size: x-small;"><i>Images: LDHA is sufficient to evoke CNS angiogenesis. (a) Representative images of CD105-labeled spinal cord sections obtained 7 days after LDHA administration. (b) Length of CD105+ neovessels around the LDHA administration site as indicated in a, n = 5 each. (c) Representative image of a Nissl-stained brain section after controlled cortical impact (CCI). (d) Representative image of the CD105-immunolabelled cerebral cortex obtained 7 days after CCI. (e) Length of CD105+ neovessels around CCI lesions as indicated in d; n = 5 each, all error bars represent the s.e.m. **P < 0.01, Student's t-tests. Scale bars, 200 μm.</i></span></div>
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The findings reveal unexpected neurovascular interactions in the injured adult CNS that may be relevant to our understanding of neuronal damage, which is a hallmark of many CNS disordersNeuromicshttp://www.blogger.com/profile/08144200376477478451noreply@blogger.com0tag:blogger.com,1999:blog-4594982080375426081.post-72008783025513184162018-01-13T09:49:00.000-08:002018-01-13T09:49:14.468-08:00Delivering siRNA in vivo<b><i>New Publications</i></b><br />
<b><i><br /></i></b> Our <a href="https://www.neuromics.com/NI35750" target="_blank">i-Fect transfection kit</a> was one of the first products. This has resulted in it being widely used and <a href="https://www.neuromics.com/transfection-kits-pubs" target="_blank">frequently published</a>.<br />
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We kick off 2018 with some new publications. Here we feature a study where researchers use our kit to deliver G-protein-coupled receptor C (MrgprC) siRNA in vivo. This receptor is part of the TRPV1 pathway.<br />
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<li>Ying-jun Liu, Xiao-xi Lin, Jian-qiao<span style="background-color: #fefefe; color: #0a0a0a; font-family: "Open Sans"; font-size: inherit;"> Fang, and Fang Fang. </span><a href="http://scholar.google.com/scholar_url?url=http://downloads.hindawi.com/journals/ecam/aip/9102107.pdf&hl=en&sa=X&scisig=AAGBfm2xCfxJ8U22kCaTBvdeUU_yN0SQuA&nossl=1&oi=scholaralrt" style="box-sizing: inherit; color: #3256a5; font-family: "Open Sans"; font-size: inherit; line-height: inherit; text-decoration-line: none;">Involvement of MrgprC in electroacupuncture analgesia for attenuating CFA-induced thermal hyperalgesia by suppressing the TRPV1 pathwa</a><span style="background-color: #fefefe; color: #0a0a0a; font-family: "Open Sans"; font-size: inherit;">y. Pre-pub-full text PDF</span></li>
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhFsoZA46mD31xdCD_hZIJh8fSmxag86rt0iIcVSkZcoZaPG4RUgSxqr95WfBJzifDcEx7iNP_ecymBcb1212kqJZjVeEvuC8cY2aMUpIouucjtPzcIHxcJQtf6Ww-yT8-AI2iDg_J1aj4/s1600/TRPV1-i-fect.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="486" data-original-width="718" height="270" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhFsoZA46mD31xdCD_hZIJh8fSmxag86rt0iIcVSkZcoZaPG4RUgSxqr95WfBJzifDcEx7iNP_ecymBcb1212kqJZjVeEvuC8cY2aMUpIouucjtPzcIHxcJQtf6Ww-yT8-AI2iDg_J1aj4/s400/TRPV1-i-fect.jpg" width="400" /></a></div>
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<span style="color: #0a0a0a; font-family: Open Sans;"><i>Image: Expression of TRPV1 in rats treated with MrgrprC si RNA vs controls (C).</i></span></div>
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<span style="color: #0a0a0a; font-family: Open Sans;">The others conclude that MrgprC expression is impacted by electroacupuncture and downregulates TRPV1. This is the mechanism that results in pain relief.</span></div>
Neuromicshttp://www.blogger.com/profile/08144200376477478451noreply@blogger.com0tag:blogger.com,1999:blog-4594982080375426081.post-25472691989048092522017-09-21T08:31:00.000-07:002017-09-21T08:31:02.253-07:00i-Fect Deliver Plasmids to the CNS<i><b>Important for Gene Expression Studies.</b></i><br />
I have posted many examples of how our customers use <a class="" href="http://www.neuromics.com/ittrium/visit/A1x66x1y1x85b1x1x9cy1x622fx1x96y1x11678x1x8ey1x11ac4x1x8ey1x351dx1x82" target="_blank">i-Fect</a><sup><a href="http://www.neuromics.com/ittrium/visit/A1x66x1y1x85b1x1x9cy1x622fx1x96y1x11678x1x8ey1x11ac4x1x8ey1x351dx1x82" target="_blank">TM </a> </sup>and other <a href="http://www.neuromics.com/gene-expression-editing-extraction-purification-and-integration-tools" target="_blank">Transfection Solutions</a> for Gene Manipulation Studies. There are also many <a href="http://www.neuromics.com/ittrium/visit/A1x66x1y1x85b0x1x9dy1xe3x1y1xd0ex1y1xd26x1" target="_blank">publications</a>.<br />
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Here we feature how i-Fect was used to delivery plasmids to the CNS: Sara Elramah, María José López-González, Matthieu Bastide, Florence Dixmérias, Olivier Roca-Lapirot, Anne-Cécile Wielanek-Bachelet, Anne Vital, Thierry Leste-Lasserre, Alexandre Brochard, Marc Landry & Alexandre Favereaux. <a href="https://www.nature.com/articles/s41598-017-10224-1" target="_blank">Spinal miRNA-124 regulates synaptopodin and nociception in an animal model of bone cancer pain</a>. Scientific Reports 7, Article number: 10949 (2017) doi:10.1038/s41598-017-10224-1...Intrathecal administration of miRNAs and ShRNA To over-express miR-124, we cloned the pre-miRNA sequence of miR-124 into a plasmid. To determine cells expressing this miR-124 encoding plasmid, we added a GFP-coding sequence to the construct under the control of an IRES. Thus, miR-124 over-expressing cells also express GFP. To inhibit synaptopodin expression, we cloned a ShRNA sequence directed against synaptopodin into a plasmid. To determine cells expressing this ShRNA, we added a GFP-coding sequence to the construct under the control of an IRES. Thus, ShRNA expressing cells also expressed GFP. Two micrograms of these plasmids or the corresponding controls, were solubilized in 10 µl of i-Fect reagent (Neuromics, Edina, USA), and injected intrathecally between the L5 and L6 lumbar vertebrae every two days for a total of 3 injections, according to the manufacturer’s instructions and previously published experiments...<br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh1fdh20pEPLWtxp4U31Js0Q9l-Ntiuyn61QKlYT6BcoMvJF_T100-UDBHQkQ6e9G-_XemjV5Y3nx_IBWTZxppifq6Ujr21nVyFQoqNL9PMe96qmF7MKnZv6zJKPtb2muzqM3qZqK5LDjk/s1600/iFect1.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" data-original-height="427" data-original-width="893" height="191" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh1fdh20pEPLWtxp4U31Js0Q9l-Ntiuyn61QKlYT6BcoMvJF_T100-UDBHQkQ6e9G-_XemjV5Y3nx_IBWTZxppifq6Ujr21nVyFQoqNL9PMe96qmF7MKnZv6zJKPtb2muzqM3qZqK5LDjk/s400/iFect1.jpg" width="400" /></a></div>
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Figures: (C and D) Immunostaining of synpo in spinal cord after miR-124 intrathecal injections: only the dorsal horn which receive nociceptive information was quantified (white dash area). Measurement of synaptopodin stained area reveals ability of miR-124 to inhibit endogenous Synpo expression (20/3 and 17/3 denotes number of sections/animals for control and miR-124-injected mice, respectively.</div>
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I am confident there will be many more positive reports regarding our Transfection Reagents.</div>
Neuromicshttp://www.blogger.com/profile/08144200376477478451noreply@blogger.com0tag:blogger.com,1999:blog-4594982080375426081.post-26570595856598163232017-01-31T12:15:00.001-08:002017-01-31T12:19:11.336-08:00Desperately Seeking Data<b><i>Answering the Bell</i></b><br />
We continue to seek data using our <a href="http://www.neuromics.com/cells-cell-based-assay-kits-models-reagents-and-media" target="_blank">cells</a>. We offer a reward of 25 USD Starbucks' Gift Card.<br />
<br />
We were pleased to receive a recently published study from Dr. Mahendran Subramanian of Keele University. In this study, researchers showed that oscillating nanomagnetic gene transfection could be used to successfully transfect SH‐SY5Y cells as well as <a href="http://www.neuromics.com/primary-neurons-astrocytes-glia-and-schwann-cells" target="_blank">our primary hippocampal and cortical neurons</a> on different days in vitro. This novel technique was used to effectively deliver genetic material into various cell types, resulting in high transfection efficiency and viability.<a href="http://www.neuromics.com/site/special/A8x12214x8x1.pdf" target="_blank"> Mahendran Subramanian, Aimee‐Jayne Tyler, Eva Maria Luther, Elena Di Daniel, Jenson Lim and Jon Dobson. Oscillating Magnet Array−Based Nanomagnetic Gene Transfection: A Valuable Tool for Molecular Neurobiology Studies</a>. Nanomaterials 2017, 7, 28; doi:10.3390/nano7020028...Primary rat hippocampal and cortical neurons were obtained from Neuromics (Edina, MN, USA) and disassociated using papain disassociation kit (Worthington, NJ, USA) according to the manufacturer’s instructions. Isolated neurons were maintained using neurobasal medium supplemented with 5% FBS, 0.5 mM Glutamax, 2% B27 supplement, 25 μM L‐glutamine and seeded onto poly‐D‐lysine–coated cells culture plates...<br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh4bAGeI4nmrg2BaWM-iI82sXHoVOMrkvUJx0DJ3dyn-ndHPvbtaFiEjwDAjfkH7u68gAs7OwZKf1K29Fp4BLq1aLRFtvS2UlxSSZZ71bQ9hEool2OS4MOMNvo1IAMt2veHf_cGfN7bPfM/s1600/Nanotherics.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="221" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh4bAGeI4nmrg2BaWM-iI82sXHoVOMrkvUJx0DJ3dyn-ndHPvbtaFiEjwDAjfkH7u68gAs7OwZKf1K29Fp4BLq1aLRFtvS2UlxSSZZ71bQ9hEool2OS4MOMNvo1IAMt2veHf_cGfN7bPfM/s400/Nanotherics.png" width="400" /></a></div>
<span style="font-size: x-small;">Figure 1. Oscillating magnet array−based nanomagnetic gene transfection experimental setup. (A) Representation of a 96‐well oscillating magnet array–based nanomagnetic transfection setup using NdFeB magnetic array (nanotherics); (B) Dimensions of the permanent magnets and magnetostatic (vectorpotential) algorithm based magnetic field density |B| distribution (T) contour plot for the NdFeB magnetic array.</span><br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhqNK_hFK_g9d8PJmk1eaIHjRzVxt4ZUfTfc1AuRpqnq9cSVi74q8ZJtw-XjEfrV-9tSnEnn-wiNNGPGAjr_yg4S5SnERE_6vnrmXQROsAeSAv8nCBQauzNh3TlTnkFqQ0vdOWYv8YfsPc/s1600/Neurons.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="172" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhqNK_hFK_g9d8PJmk1eaIHjRzVxt4ZUfTfc1AuRpqnq9cSVi74q8ZJtw-XjEfrV-9tSnEnn-wiNNGPGAjr_yg4S5SnERE_6vnrmXQROsAeSAv8nCBQauzNh3TlTnkFqQ0vdOWYv8YfsPc/s400/Neurons.png" width="400" /></a></div>
<span style="font-size: x-small;"><br /></span><span style="font-size: x-small;"> Figure 2. Gene delivery by oscillating nanomagnetic gene transfection in primary cortical neurons. Images of pmaxGFP plasmid expressed in primary neurons using fluorescence microscopy and its corresponding Hoechst 33,342 stained counterpart of transfected DIV 1 (A,C) and DIV 5 (B,D) mature neurons were taken 48 h post transfection.</span><br />
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If you have data to share email it to me, <a href="mailto:pshuster@neuromics.com">pshuster@neuromics.com</a> and we'll email you a 25 USD gift card. Thank you. Pete Shuster, CEO & Owner.Neuromicshttp://www.blogger.com/profile/08144200376477478451noreply@blogger.com0tag:blogger.com,1999:blog-4594982080375426081.post-60911442455498097402017-01-30T05:22:00.001-08:002017-01-30T05:23:46.267-08:00More iFect in-vivoThe parade of publications continues to grow.<br />
<br />
Here researchers use our<a href="http://www.neuromics.com/ittrium/visit/A1x66x1y1x85b1x1x9cy1x622fx1x96y1x11678x1x8ey1x11ac4x1x8ey1x351dx1x82" target="_blank"> i-Fect<sup>TM</sup> Transfection Kit</a> for delivering sh-IRF3 <i>in vivo</i>: <a href="http://www.sciencedirect.com/science/article/pii/S0753332216323150" target="_blank">Rui Li, Li-guo Wang, Qi Wang, Zhi-hua Li, Ya-li Ma, Qing-Duo Guo. Silencing of IRF3 alleviates chronic neuropathic pain following chronic constriction injury</a>. doi.org/10.1016/j.biopha.2017.01.085... The oligonucleotides for sh-IRF3 were: 5′-CACCGCGTCTAGGCTGGTGGTTATTCGAAAATAACCACCAGCCTAGACGC-3′ −3′. Then, 10 μg sh-IRF3 dissolved in 30 μl i-Fect transfection reagent (Neuromics, Edina, MN, USA) was administered intrathecally once daily for 7...<br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhe1HDdizxj3vD8sfzlrUr1knA4IZNT3qRcQTagUl3STTRziH57eFSc_1dNkMoWMAWg6L4wWCxOR-pwZ7mzLm1tXyrRaZIor0RLOMXqLb7rjXF_rpQOKNYSiG5zlBjvoDdZKB4nOzsirXQ/s1600/IFR3_i-Fect.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhe1HDdizxj3vD8sfzlrUr1knA4IZNT3qRcQTagUl3STTRziH57eFSc_1dNkMoWMAWg6L4wWCxOR-pwZ7mzLm1tXyrRaZIor0RLOMXqLb7rjXF_rpQOKNYSiG5zlBjvoDdZKB4nOzsirXQ/s400/IFR3_i-Fect.jpg" width="307" /></a></div>
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<span style="font-size: x-small;"><i>Fig. Down-regulation of IRF3 attenuated mechanical allodynia and thermal hyperalgesia in CCI rats. (A) The mRNA expression level of IRF3 in the DRG at postoperative day 7. (B) The protein expression level of IRF3 in the DRG at postoperative day 7. (C and D) PWT and PWL were measured 1 day before CCI and 1, 3, 7 and 14 days after intrathecal injection of sh-IRF3 or scramble</i></span>.</div>
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Down-regulation of IRF3 inhibited the production of pro-inflammatory cytokines in the DRG of CCI rats.<br />
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These results indicated that IRF3 was involved in the development of neuropathic pain. Down-regulation of IRF3 attenuated neuropathic pain in CCI rats by inhibiting the activation of NF-κB signaling pathway, suggesting that IRF3 may be a novel and potential target for the treatment of neuropathic pain.Neuromicshttp://www.blogger.com/profile/08144200376477478451noreply@blogger.com0tag:blogger.com,1999:blog-4594982080375426081.post-90608074429548173592016-08-19T05:36:00.001-07:002016-08-19T05:36:21.016-07:00Delivering miRNA in vivo<b>More Transfection Success!</b>
<b>Great Research Tools!</b><br />
<b><br /></b>
Our <a href="http://www.neuromics.com/ittrium/visit/A1x66x1y1x85b0x1x9dy1xe3x1y1xd0ex1y1xd26x1" target="_blank">i-Fect<sup>TM<b> </b></sup> Transfection Ki</a>t is used to study <a href="http://www.neuromics.com/ittrium/visit/A1x66x1y1x85b1x1x9cy1x622fx1x96y1x11678x1x8ey1x11ac4x1x8ey1x351dx1x82" target="_blank">Epigenetics</a> and pain. Here's yet another example: <a href="http://www.sciencedirect.com/science/article/pii/S001448861630190X" target="_blank">M. Leinders, b, N. Üçeyler, R.A. Pritchard, C. Sommer, L.S. Sorkin. Increased miR-132-3p expression is associated with chronic neuropathic pain</a>. Experimental Neurology. Volume 283, Part A, September 2016, Pages 276–286...The inhibitor and mimetic were administered to awake rats via the it catheters. Prior to injection, active or mismatch inhibitors were mixed with (1:5 w/v) i-Fect™ in vivo transfection reagent(Neuromics, Edina, USA) to final doses of 5, 2 and 1 μg in 10 μl...<br />
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Spinal administration of miR-132-3p antagonists via intrathecal (i.t.) catheters dose dependently reversed mechanical allodyina and eliminated pain behavior in the place escape avoidance paradigm (p < 0.001). Intrathecal administration of miR-132-3p mimetic dose-dependently induced pain behavior in naïve rats (p < 0.001). Taken together these results indicate a pro-nociceptive effect of miR-132-3p in chronic neuropathic pain.<br />
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Finding like these could pave the way for an miRNA like therapy for pain.Neuromicshttp://www.blogger.com/profile/08144200376477478451noreply@blogger.com0tag:blogger.com,1999:blog-4594982080375426081.post-50284974883187925052016-06-20T08:21:00.000-07:002016-06-20T08:21:17.695-07:00i-Fect used to Study Epigenetics in Nerve Injury<b><i>i-Fect used In Vivo for Study</i></b><br />
<b><i><br /></i></b>
Researchers use our <a href="http://www.neuromics.com/ittrium/visit/A1x66x1y1x85b1x1x9cy1x622fx1x96y1x11678x1x8ey1x11ac4x1x8ey1x351dx1x82" target="_blank">i-Fect<sup>TM</sup></a> to effectively deliver miR-126 <i>in vivo </i>to modulate Methyl-CpG-binding protein 2 (MeCP2).<br />
<br />
MeCP2 regulates gene expression through activation, repression and chromatin remodeling. Mutations in MeCP2 cause Rett syndrome, and these patients display impaired nociception. <a href="http://epigeneticsandchromatin.biomedcentral.com/articles/10.1186/s13072-016-0073-5" target="_blank">The researchers observed an increase in MeCP2 expression in mouse dorsal root ganglia (DRG) after peripheral nerve injury: Melissa T. Manners, Adam Ertel, Yuzhen Tian and Seena K. Ajit. Genome-wide redistribution of MeCP2 in dorsal root ganglia after peripheral nerve injury</a>. Epigenetics & Chromatin 20169:23. DOI: 10.1186/s13072-016-0073-5© The Author(s) 2016 Received: 11 March 2016. Accepted: 27 May 2016Published: 7 June 2016...miRNA administration protocol was adapted from previous report of intrathecal miRNA delivery. To administer miRNA mimics, a polyurethane catheter (25G, 5.5 cm long, SAI infusion) was placed into the intrathecal space of the lumber L4–L5 vertebrae under isoflurane anesthesia. The catheter was stereotactically secured under the skin and occluded between injections. A custom miRCURY (Exiqon) miR-126 mimic containing a 5′ cholesterol tag and 3′ fluorescein label was injected at 2 nmol concentration with 4 µl <a href="http://www.neuromics.com/ittrium/visit/A1x66x1y1x85b1x1x9cy1x622fx1x96y1x11678x1x8ey1x11ac4x1x8ey1x351dx1x82" target="_blank">iFECT </a>transfection reagent (Neuromics). A total of 6 µl was delivered into the catheter connection juncture using a 25G blunt end needle on a Hamilton syringe. The catheter was then flushed with 7 µl sterile PBS to ensure miRNA reached the intrathecal space...<br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg8CGb28I5BNJJfm7XbMw1JpUsTTDyhjGMu9OL_FzW1e7nof-6NFkhIrRO1wz6vFkPkVlI1xzGcDy5Zao9ECjgT0C2EJSbfpcCnQg4RpOvgAHjHKbjlDXnPMx_JBw3BqBmJhlan8XCebSFr/s1600/i-FECT-MM-drg.gif" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="291" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg8CGb28I5BNJJfm7XbMw1JpUsTTDyhjGMu9OL_FzW1e7nof-6NFkhIrRO1wz6vFkPkVlI1xzGcDy5Zao9ECjgT0C2EJSbfpcCnQg4RpOvgAHjHKbjlDXnPMx_JBw3BqBmJhlan8XCebSFr/s400/i-FECT-MM-drg.gif" width="400" /></a></div>
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<span style="font-size: x-small;">Figure: Expression of miR-126 and its target genes Dnmt1 and Vegfa in the DRG after nerve injury. a Relative expression of miR-126 determined by qPCR shows a reduction in miR-126 in SNI model compared to DRG from sham control. U6 was used for normalization (n = 8 sham, n = 7 SNI). b Relative expression of Dnmt1 mRNA and c Vegfa transcripts showed an increase in the DRG after nerve injury compared to control (n = 3). Gapdh was used as a normalizer. d Representative Western blot and quantification showed an increase of Dnmt1 protein in the DRG after nerve injury. e Western blot and quantification showed Vegfa protein was not significantly different in DRG after nerve injury (n = 3 from pooled samples, three DRG were pooled for each sample).</span></div>
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<b>Conclusions:</b> The study shows a regulatory role for MeCP2 in that changes in global redistribution can result in direct and indirect modulation of gene expression in the DRG. Alterations in genome-wide binding of MeCP2 therefore provide a molecular basis for a better understanding of epigenetic regulation-induced molecular changes underlying nerve injury.Neuromicshttp://www.blogger.com/profile/08144200376477478451noreply@blogger.com0tag:blogger.com,1999:blog-4594982080375426081.post-70414997428404616252016-03-24T09:18:00.000-07:002016-03-24T09:18:28.635-07:00Epigenetics and Pain Research<b><i>i-Fect Used to Study Impacts</i></b><br />
<br />
Our <a href="http://www.neuromics.com/ittrium/visit/A1x66x1y1x85b1x1x9cy1x622fx1x96y1x11678x1x8ey1x11ac4x1x8ey1x351dx1x82" target="_blank">i-Fect siRNA, miRNA and shRNA Trasfection Kit</a> was recently used to study the impact of G9a-specific siRNA
(AGUAACGGGCAUCAAUGC) on Mu Opioid Receptors:<a href="http://www.jbc.org/content/early/2016/02/25/jbc.M115.711812.full.pdf+html" target="_blank"> Yuhao Zhang, Shao-Rui Chen, Geoffroy Laumet, Hong Chen and Hui-Lin Pan. Nerve Injury Diminishes Opioid Analgesia through Lysine Methyltransferase-Mediated Transcriptional Repression of µ-Opioid Receptors in Primary Sensory Neurons.</a> First Published on February 25, 2016, doi: 10.1074/jbc.M115.711812... In some SNL rats, G9a-specific siRNA (4 µg) or the negative control siRNA was administered intrathecally. G9a-specific siRNA(AGUAACGGGCAUCAAUGC) or universal negative control siRNA (#SIC001, Sigma-Aldrich) was mixed with i-Fect (Neuromics, Edina, MN) to a final concentration of 400 mg/L for the intrathecal injections...
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhw1T3ue6SPkRZS0KBASnKQHdpLIEprvRuyZbGjzM1RFFUpeCB21sGiN_r-ENErd6V7REq_T6uTy5hLOgmdo6hqEMyYol5dyxrZpExPgvGnKuTfryhQgEljFst7byYz7PuEiyKNKRPOquA/s1600/iFect_MOR.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhw1T3ue6SPkRZS0KBASnKQHdpLIEprvRuyZbGjzM1RFFUpeCB21sGiN_r-ENErd6V7REq_T6uTy5hLOgmdo6hqEMyYol5dyxrZpExPgvGnKuTfryhQgEljFst7byYz7PuEiyKNKRPOquA/s400/iFect_MOR.jpg" width="363" /></a></div>
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<span style="font-size: x-small;"><i>Figures: G9a knockdown with siRNA reverses the MOR expression in the DRG and the morphine analgesic
effect diminished by nerve injury. (A,B) Quantitative PCR (A) and Western blotting (B) analyses show the mRNA
and protein levels of MORs in the DRGs of sham and SNL rats treated with control or G9a-specific siRNA (n = 10
rats in each group). The ipsilateral L5 and L6 DRG tissues were removed 24 h after the last siRNA injection. The
amount of MOR mRNA and protein was normalized to GAPDH in the same samples, and the mean value of MOR
levels in sham control rats was considered to be 1. (C) Time course of the intrathecal morphine effects on the tactile
and pressure withdrawal thresholds in sham and SNL rats treated with G9a-specific siRNA or negative control
siRNA (n = 9 rats in each group). The withdrawal thresholds after the last siRNA injection were plotted as the
baseline control (BL). </i></span><br />
<span style="font-size: x-small;"><i><br /></i></span>
<span style="font-size: x-small;"><i><br /></i></span><b>
Summary:</b> The findings provide new insight
into the epigenetic mechanism regulating MOR
expression in primary sensory neurons in neuropathic
pain. This multidisciplinary approach provides
conclusive evidence for G9a as a key chromatin
regulator responsible for MOR downregulation in the
DRG and the analgesic efficacy of opioids reduced by
nerve injury. A better understanding of the epigenetic
mechanisms underlying nerve injury-induced
downregulation of MORs in primary sensory neurons
could help improve the analgesic efficacy of opioids for
treating chronic neuropathic pain. G9a inhibitors could
be used to enhance the opioid analgesic effect and
reduce opioid consumption in patients with chronic
neuropathic pain. Neuromicshttp://www.blogger.com/profile/08144200376477478451noreply@blogger.com0tag:blogger.com,1999:blog-4594982080375426081.post-73408986867655985732016-01-20T07:02:00.000-08:002016-03-24T07:42:44.669-07:00Delivery of siRNA, miRNA, shRNA and Plasmids GuaranteedPotent and Frequently Published Transfection Solutions
<br />
<br />
<strong style="text-align: center;"><em><a href="http://www.neuromics.com/gene-expression-editing-and-integration-tools" title="Gene Tools"><img alt="Gene Tools" src="http://logon.salesnexus.com/mIscref/Neuromics/images/SB_Web.jpg" height="61" title="Gene Tools" width="61" /></a></em></strong><br />
<span class="style18"><span class="style16">We have proven and frequently <a href="http://www.neuromics.com/ittrium/visit/A1x66x1y1x85b0x1x9dy1xe3x1y1xd0ex1y1xd26x1"> published transfection solutions</a>. </span></span><br />
<span class="style18"><span class="style16">Powerful punch and versatility are now needed more than ever with the </span></span><br />
<span class="style18"><span class="style16">hyper growth in gene manipulation technologies like <a href="http://www.neuromics.com/ittrium/visit/A1x66x1y1x85b1x1x9cy1x622fx1x96y1x11678x1x8ey1x11ac5x1x8ey1xb226x1x82"> Sleeping Beauty<sup>TM</sup></a></span></span><br />
and CRISPR-Cas9.<br />
<span class="style18"><span class="style16"><br /><span style="font-size: xx-small;">We have published examples of the use of our solutions for delivering siRNA, miRNA, </span></span></span><br />
<span class="style18"><span class="style16"><span style="font-size: xx-small;">shRNA and </span></span></span>plasmids both <em>in vitro</em> and <em>in vivo. h</em>ere are some of your options<em style="font-size: small;">.</em><br />
<span style="font-size: x-small;"><a href="http://www.neuromics.com/ittrium/visit/A1x66x1y1x85b1x1x9cy1x622fx1x96y1x11678x1x8ey1x11ac4x1x8ey1x351dx1x82" title="A novel cationic lipid formulation specifically designed for efficient delivery of 27mer DsiRNAs(dicer substrate small Interfering RNAs)& 21mer siRNAs (small interfering RNAs) in vitro and in vivo."> i-Fect ™</a> -A novel cationic lipid formulation specifically </span><br />
<span style="font-size: x-small;">designed for efficient delivery of 27mer DsiRNAs(dicer </span><br />
<span style="font-size: x-small;">substrate small Interfering RNAs)& 21mer siRNAs (small interfering RNAs) in vitro and in vivo.</span><br />
<span style="font-size: x-small;"><a href="http://www.neuromics.com/ittrium/visit/A1x66x1y1x622fx1x96y1x5118x1x82">p-Fect™</a> -Designed to delivery plasmids, DNA or </span><br />
<span style="font-size: x-small;">RNA to hard to transfect Cell Lines.</span><br />
<div class="auto-style2">
</div>
<span style="font-size: x-small;"><a href="http://www.neuromics.com/ittrium/visit/A1x66x1y1x622fx1x96y1x3524x1x82">pn-Fect™</a> -The latest advance in transfection </span><br />
<span style="font-size: x-small;">for primary neuronal cells. </span><br />
<span style="font-size: x-small;">This unique reagent provides ultra-high plasmid DNA delivery </span><br />
<span style="font-size: x-small;">efficiencies and low cytotoxicity compared to competitive reagents.</span><br />
<span style="font-size: xx-small;">Here's a recent i-Fect Publication:</span><br />
<a href="http://link.springer.com/article/10.1007/s12031-016-0713-z" target="_blank" title="i-Fect Customer Publication"> Liuming Jiang, Qun Wu , Tao Yang. Silencing of Id2 Alleviates </a><br />
<a href="http://link.springer.com/article/10.1007/s12031-016-0713-z" target="_blank" title="i-Fect Customer Publication">Chronic Neuropathic Pain Following Chronic Constriction Injury</a>.<br />
Journal of Molecular Neuroscience\pp 1-7.First online: 15 January 2016<br />
<a href="http://www.neuromics.com/ittrium/visit/A1x66x1y1x85b1x1x9cy1x622fx1x96y1x11678x1x8ey1x11ac4x1x8ey1x351dx1x82" title="i-Fect Transfection Kit"><img alt="i-Fect" src="http://logon.salesnexus.com/mIscref/Neuromics/images/i-Fect_ID2.jpg" height="458" title="i-Fect" width="437" /></a><br />
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<em>Figure: Knockdown of Id2 attenuated mechanical allodynia and thermal </em></div>
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<em>hyperalgesia in CCI rats. (a and b) PWT and PWL were measured 1 day before </em></div>
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<em>CCI and 1, 3, 7, and 14 days after intrathecal administration of shRNA-Id2. </em></div>
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<span class="style11">If you are looking for transfection solutions, do not hesitate to contact me @ direct phone: 612-801-1007 or <a href="mailto:pshuster@neuromics.com">pshuster@neuromics.com</a>. Thank you, Pete Shuster, CEO and Owner, Neuromics</span><strong><span class="style11"><br /></span></strong></div>
Neuromicshttp://www.blogger.com/profile/08144200376477478451noreply@blogger.com0tag:blogger.com,1999:blog-4594982080375426081.post-48172684706637777112015-11-16T12:07:00.004-08:002015-11-16T12:07:20.394-08:00Kv Channels and Acute to Chronic Pain Transistioni-Fect <sup>TM</sup>is a Proven Tool for Gene Manipulation in Studying All Types of Pain<br />
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I previously posted on use of our <a href="http://www.neuromics.com/ittrium/visit/A1x66x1y1x85b1x1x9cy1x622fx1x96y1x351dx1x82" target="_blank">i-Fect Transfection Kit</a> to silence Kv Channels Receptors. This has enabled researchers to study the role of these receptors <i>in vitro</i> and <i>in vivo (</i>see:i-Fect™ Delivers Your siRNA Payload<i>).</i><br />
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<i><b>Sample Data</b></i><br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjTJgPJ19jDLB-izPe12sk4F50EYGXac03paPga9G9bjtg7sgAVKipSRkdQH62jbfyGU436axT8BkoxRKeN40aV0rV0UbkfSpC5lJvW4as4eYVgeSOIueztIOz-IhubAIrl6l0sZyQTjTQ/s1600/Kv1.1-siRNA+%25281%2529.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="262" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjTJgPJ19jDLB-izPe12sk4F50EYGXac03paPga9G9bjtg7sgAVKipSRkdQH62jbfyGU436axT8BkoxRKeN40aV0rV0UbkfSpC5lJvW4as4eYVgeSOIueztIOz-IhubAIrl6l0sZyQTjTQ/s400/Kv1.1-siRNA+%25281%2529.jpg" width="400" /></a></div>
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<i><span style="font-size: x-small;">Figure: 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</span></i></div>
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I am pleased to share with you a new reference detailing how research use i-Fect to optimize and deliver euchromatic histone-lysine N-methyltransferase-2 (G9a) siRNA. This brings the number of <a href="http://www.neuromics.com/ittrium/visit/A1x66x1y1x85b0x1x9dy1xe3x1y1xd0ex1y1xd26x1" target="_blank">publications</a> referencing use of our <a href="http://www.neuromics.com/transfection-kits-and-reagents" target="_blank">Transfection Kits</a> to over 45: <a href="http://www.nature.com/neuro/journal/vaop/ncurrent/full/nn.4165.html" target="_blank">Geoffroy Laumet, Judit Garriga, Shao-Rui Chen, Yuhao Zhang, De-Pei Li, Trevor M Smith, Yingchun Dong, Jaroslav Jelinek, Matteo Cesaroni, Jean-Pierre Issa & Hui-Lin Pan G9a is essential for epigenetic silencing of K+channel genes in acute-to-chronic pain transition.</a> Nature Neuroscience (2015) doi:10.1038/nn.4165.<br />
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The authors report: "Selective knockout of the gene encoding G9a in DRG neurons completely blocked K+ channel silencing and chronic pain development after nerve injury. Remarkably, RNA sequencing analysis revealed that G9a inhibition not only reactivated 40 of 42 silenced genes associated with K+ channels but also normalized 638 genes down- or upregulated by nerve injury."<br />
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I will continue to post here new and unique solutions and related referencing for our Gene Expression Analysis Tools.</div>
Neuromicshttp://www.blogger.com/profile/08144200376477478451noreply@blogger.com0tag:blogger.com,1999:blog-4594982080375426081.post-23433055376015791162015-08-03T14:01:00.000-07:002015-08-03T14:03:25.889-07:00Introducing Sleeping Beauty<strong><em>New Technology for Gene Transfer</em></strong>
<strong><em>From Delivery to Stable Expression</em></strong><br />
Neuromics has a successful track record of helping our clients delivery siRNA, miRNA, Plasmids and other oligos in vitro and in vivo with our <a href="http://www.neuromics.com/ittrium/visit/A1x66x1y1x85b0x1x9dy1xe3x1y1xd0ex1y1xd26x1" target="_blank">Transfection Kits</a>...But my vision with our <a href="http://www.neuromics.com/cells-cell-based-assay-kits-reagents-and-media" target="_blank">cell based assay solutions</a> has always been to provide engineered cells and plasmids modified to study your genes of interest. I am pleased to announce we are working with Smart Cell /B-MoGen Technologies to make this happen.
We now can provide:<br />
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Gene Transfer and Expression Products
Leveraging the <em>Sleeping Beauty</em> Technology</div>
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh2fT4Vi1N5w2OtFLyY2ZDt_sqcV910WbtCVyhsPiQXigE2F2Rm4QeOdhHoGydpj40kU0zbjsKHtd_yD_75fpEYNVQ6Sy29fVQEiWm97eI9Z7whwfVvEeUwAFBA0ozD084gLnBNQqMda9Tp/s1600/B_Mogen1.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="228" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh2fT4Vi1N5w2OtFLyY2ZDt_sqcV910WbtCVyhsPiQXigE2F2Rm4QeOdhHoGydpj40kU0zbjsKHtd_yD_75fpEYNVQ6Sy29fVQEiWm97eI9Z7whwfVvEeUwAFBA0ozD084gLnBNQqMda9Tp/s400/B_Mogen1.jpg" width="400" /></a></div>
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<em>Images: B-MoGen Transposon exhibiting stable expression of five fluorescent genes</em>.
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Advantages of Sleeping Beauty Transposon System:
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· Delivery method is time and cost effective compared to lentiviral delivery.
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· Increased cargo-capacity when compared to lentiviral delivery.
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· Safest insertion profile of all gene transfer methods.
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· Commonly integrated as a single copy.
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Custom vector design and assembly, including multi-gene (up to 6) vectors.
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We are in the process of formulating standard offerings. In the meantime, I am positioned to offer favorable pricing and terms to early adopters of our Sleeping Beauty Solutions. Please contact me directly <a href="mailto:pshuster@neuromics.com">pshuster@neuromics.com</a> or 612-801-1007. We can together determine your needs and desired outcomes and provide a statement of work with pricing, project milestones and delivery.Neuromicshttp://www.blogger.com/profile/08144200376477478451noreply@blogger.com0tag:blogger.com,1999:blog-4594982080375426081.post-90606627052158860632015-04-07T16:39:00.000-07:002015-05-11T10:48:19.995-07:00Silencing Cytokines in-vivo with i-Fect<b><i>Knocking Down Cytokines to Study Pain Response</i></b><br />
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We have many unique applications <a href="http://www.neuromics.com/ittrium/visit/A1x66x1y1x85b0x1x9dy1xe3x1y1xd0ex1y1xd26x1" target="_blank">published</a> by researchers using our <a href="http://www.neuromics.com/transfection-kits-and-reagents" target="_blank">Transfection Kits</a> <i>in vitro and in vivo. </i>Here researchers simultaneously silence 3 immune/inflammatory response cytokines <i>in vivo</i>: <a href="http://synapse.koreamed.org/DOIx.php?id=10.4097/kjae.2015.68.2.159" target="_blank">Byung Moon Choi, Soo Han Lee, Sang Mee An, Do Yang Park, Gwan Woo Lee, and Gyu-Jeong Noh. The time-course and RNA interference of TNF-α, IL-6, and IL-1β expression on neuropathic pain induced by L5 spinal nerve transection in rats</a>. Korean J Anesthesiol. 2015 Apr;68(2):159-169. English. Published online March 30, 2015. http://dx.doi.org/10.4097/kjae.2015.68.2.159.<br />
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<b>Protocol: </b>RNAs were administered as described, with modifications [11]. A cocktail of siRNA simultaneously targeting TNF-α (Silencer® Select siRNA; s128522, Ambion, Austin, TX, USA), IL-6 (Silencer® Select siRNA; s217844, Ambion, Austin, TX, USA) and IL-1b (Silencer® Select siRNA; s127941, Ambion, Austin, TX, USA), as well as a control siRNA (Silencer® Negative Control #1 siRNA; Cat #4635, Ambion, Austin, TX, USA), were prepared immediately prior to administration by mixing the RNA (200 µM) with the transfection reagent, <a href="http://www.neuromics.com/ittrium/visit/A1x66x1y1x85b1x1x9cy1x622fx1x96y1x351dx1x82" target="_blank">i-Fect™</a> (Neuromics, Minneapolis, MN, USA), in a ratio of 1 : 5 (w : v). At this ratio, the final RNA/lipid complex concentration was 2 µg in 5 µl for each cytokine siRNA and 6 µg in 15 µl for the control siRNA. The cytokine siRNAs were combined and they and the control siRNA (15 µl each) were delivered to the lumbar region of the spinal cord via the intrathecal catheters. Injections were given daily on 5 consecutive days (-1, 0, 1, 2, 3 d after L5 SNT.<br />
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The changes in mechanically induced allodynia and hyperalgesia in the rats surviving for 6 d after SNT are shown in figure. Allodynia and hyperalgesia were lower in the COCK group than in the CON group by 2 d after SNT (P < 0.05) and the difference was maintained for the duration of the experiment.<br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhKqDjbrcY6l17oB0rVT1GbSNqoGjaz-fBBC0wc6aXcaEKDdL5u-Hobb-yK_yxv9YB4yAzV9L3MfxShVFf0usJiecaQ2MntfTnYAfL2ezDtADoJXWWBu-ixdbNB4s2xkvXc1nNbqm74WcE/s1600/i-Fect_Pain.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="140" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhKqDjbrcY6l17oB0rVT1GbSNqoGjaz-fBBC0wc6aXcaEKDdL5u-Hobb-yK_yxv9YB4yAzV9L3MfxShVFf0usJiecaQ2MntfTnYAfL2ezDtADoJXWWBu-ixdbNB4s2xkvXc1nNbqm74WcE/s1600/i-Fect_Pain.jpg" width="400" /></a></div>
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<span style="font-size: x-small;"><i>Figure: The time course of mechanical allodynia (A) and hyperalgesia (B) in the ipsilateral hind paw of rats undergoing L5 spinal nerve transection (SNT) after the administration of control siRNA (CON group) or a cocktail of small interfering RNAs (siRNA) targeting TNF-α, IL-6 and IL1-β (COCK group). The data on the rats surviving for 6 d after SNT are expressed as mean ± SE. -1: 1 d prior to SNT, 0: the day of L5 SNT, 1, 2, 4 and 6: 1, 2, 4 and 6 d after L5 SNT. *P < 0.05 vs. CON group at each time point. MPE: maximal possible effect. The cut-off values for mechanical allodynia and hyperalgesia are 30 g and 250 g, respectively.</i></span></div>
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We will continue to post new applications and methods published by researchers using our Transfection Kits.Neuromicshttp://www.blogger.com/profile/08144200376477478451noreply@blogger.com0tag:blogger.com,1999:blog-4594982080375426081.post-12160300179836463362015-02-18T04:19:00.001-08:002015-02-21T07:32:43.656-08:00EPO Protects Against Cerebral IschemiaNeuromics' <a href="http://www.neuromics.com/ittrium/visit/A1x66x1y1x85b1x1x9cy1x622fx1x96y1x351dx1x82" target="_blank">i-Fect <sup>TM</sup></a> used to Modulate phospho-Connexin 43<br />
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In this study data suggest the protective effects of EPO on NUV injuries are highly associated with the increase of p-Cx43, which improves GJIC to reduce neurotoxic substances:<a href="http://www.sciencedirect.com/science/article/pii/S0006291X15002363" target="_blank"> Ziyi Zhoua, Xiaobai Weib, Jun Xiang, Junpeng Gao, Lixin Wang, Jinsong You, Yefeng Cai , Dingfang Caid. Protection of erythropoietin against ischemic neurovascular unit injuries through the effects of connexin43.</a> Biochemical and Biophysical Research Communications. doi:10.1016/j.bbrc.2015.02...The strands were incubated at 90°C for 5 min and then at 37°C for 1 h. SiRNA was prepared immediately before administration by mixing the RNA solution (1 μg/μl in annealing buffer) with the transfection reagent i-Fect (v/v: 1/3; Neuromics, Edina, MN, USA) to a final siRNA ...
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Highlights<br />
•EPO has protective effects on ischemic NVU injuries.<br />
•EPO up-regulates phosphorylation of Cx43, not total Cx43.<br />
•EPO's protective effects on NUV injuries are p-Cx43-GJIC dependent.Neuromicshttp://www.blogger.com/profile/08144200376477478451noreply@blogger.com0tag:blogger.com,1999:blog-4594982080375426081.post-91667049180059618782014-11-24T07:33:00.000-08:002014-11-24T07:46:05.464-08:00HDAC2 and Anxiety in Alcoholism<b><i>The Impact of HDAC2 Gene Expression on Anxiety</i></b><br />
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Our <a href="http://www.neuromics.com/ittrium/visit/A1x66x1y1x85b1x1x9cy1x622fx1x96y1x351dx1x82" target="_blank">i-Fect Transfection Kit</a> continues to be a potent tool for testing the impact of altered gene expression on behavior. see: <a href="https://dspace-prod-lib.cc.uic.edu/bitstream/handle/10027/18785/Moonat_Sachin.pdf?sequence=1" target="_blank" title="i-Fect in vivo customer publications">SACHIN MOONAT. The Role of Amygdaloid Chromatin and Synaptic Remodeling in Anxiety and Alcoholism</a>. THESIS Submitted as partial fulfillment of the requirements for the degree of Doctor of Philosophy in Neuroscience in the Graduate College of the University of Illinois at Chicago, 2014.<br />
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The author hypothesized that increased HDAC2 would have a positive impact on anxiety in alchohol preferring (P) rats. Specifically, HDAC2-induced histone modifications in the amygdala may play a role in the regulation of synaptic plasticity that may underlie the behavioral phenotypes of P rats. Furthermore, it could be possible that exogenous manipulation of HDAC2 levels in the amygdala may have an effect on anxiety-like behaviors and alcohol preference in P
rats.
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg6SeE_a75LzotzJL21HcDhXJfwXbpPqKlZgjSVMaYOkcWupqtd5F1kPgZnrwl9C4IEatSFGcw7HISLIuvDq_VK8Xm4bIP-Hyl-g9Cx9Y_Y7tXs1-M2EHC70eePBtnwGop4aQBMlxr3vN4/s1600/HDAC.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg6SeE_a75LzotzJL21HcDhXJfwXbpPqKlZgjSVMaYOkcWupqtd5F1kPgZnrwl9C4IEatSFGcw7HISLIuvDq_VK8Xm4bIP-Hyl-g9Cx9Y_Y7tXs1-M2EHC70eePBtnwGop4aQBMlxr3vN4/s1600/HDAC.jpg" height="366" width="400" /></a></div>
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Figure 1. Chromatin remodeling via histone acetylation and DNA methylation regulates gene transcription associated with changes in synaptic plasticity. During gene transcriptional processes, the chromatin structure associated with DNA to be transcribed is in a relaxed chromatin conformation due to hyperacetylation of histone proteins and hypomethylation of DNA, which allows access to transcriptional machinery. This relaxed chromatin structure results in increased gene transcription, which in neurons may cause increased expression of synaptically active proteins that result in the positive modulation of synaptic plasticity, such as increased dendritic spine density (DSD). DNA methyltransferase (DNMT) methylates DNA at CpG islands, leading to hypermethylated DNA and recruiting of methyl-CpG binding domain protein (MBD) complexes which block binding of transcriptional machinery. The MBD complex can in turn recruit histone deactylases (HDAC) which remove acetyl groups from histone proteins resulting in chromatin condensation thereby decreasing gene transcription. HDACs and histone acetyltransferases (HAT) control the histone acetylation profile, such that HDACs remove acetyl groups and HATs add acetyl groups to histone proteins. In this manner, increased HDAC expression results in hypoacetylation of histones leading to a condensed chromatin structure. Chromatin condensation resulting from HDAC-induced histone deacetylation or DNMT-induced DNA methylation causes reduced gene transcription. In neuronal cells, the reduction in gene transcription may be associated with decreased expression of synaptically active proteins and negative modulation of synaptic plasticity, such as reduced DSD. Treatment with DNMT inhibitors or HDAC inhibitors may block these enzymatic processes and return chromatin to a relaxed state, resulting in increased gene transcription and synaptic plasticity (Moonat and Pandey, 2012)</i></span>.
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Methods: P rats that had been previously cannulated for delivery of solutions directly into the CeA
were infused with either HDAC2 siRNA, control siRNA or vehicle. The siRNAs were
dissolved in iFect solution (Neuromics, Edina, MN), a cationic lipid-based transfection
solution, such that the final concentration of the solution was 2 µg/µL. The sequence of
the HDAC2 siRNA was as follows: 5’-CAAGUUUCUACGAUCAACATT-3’; 5’-
UAUUGAUCGUAGAAACUUGAT-3’. Some of the HDAC2 siRNA (Qiagen, Valencia,
CA) had been modified to include a 5’ Alexa Fluor-488 fluorescent probe in order to
determine the transfection efficiency and cellular localization of transfection. The control
siRNA used was the AllStars Negative Control siRNA (Qiagen), which shows no
homology to any known mammalian gene. To prepare the vehicle, RNase-free water was
dissolved in the iFect solution in place of any siRNA. The solutions (0.5 µL) were
infused bilaterally into the CeA of P rats using an automatic infusion pump which
resulted in a dose of 1 µg of siRNA per side. The automatic pump was attached to a
microdialysis probe which seated in the guide cannula and extended 3 mm past the tip of the cannula into the CeA.<br />
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For the experiments which looked at the anxiolytic effect of HDAC2 siRNA
infusion, P rats were infused with either HDAC2 siRNA, control siRNA or vehicle at the
end of the light cycle. 16 hours after the infusion, the rats were tested for anxiety-like behaviors. Immediately following behavioral testing, rats were anaesthetized and brains
were collected for further analysis.
For the voluntary drinking experiment, P rats were infused with either HDAC2
siRNA or vehicle when the bottles were changed following the third day of 9% ethanol
exposure. The rats continued to be monitored for the intake of 9% ethanol for 7 days
following the infusion. After the final day of voluntary drinking, the rats were
anaesthetized for collection of brains and blood to confirm the cannula position and the
blood alcohol levels, respectively. <br />
<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgZ74wVhLN41SBS51j-1xAHSMoSvsgnYMwA5uhCagjm6Uy2XVd5BD23yTPG-PF7swu_P-AdpVqyEpa39AOh3LSeI47dw_c_EzShZ3oXqtqXpe4m8EWC5VAqjJYxFoA4rz3kz2kEO6zm1vw/s1600/HDAC2_P.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em; text-align: center;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgZ74wVhLN41SBS51j-1xAHSMoSvsgnYMwA5uhCagjm6Uy2XVd5BD23yTPG-PF7swu_P-AdpVqyEpa39AOh3LSeI47dw_c_EzShZ3oXqtqXpe4m8EWC5VAqjJYxFoA4rz3kz2kEO6zm1vw/s1600/HDAC2_P.jpg" height="180" width="400" /></a><br />
<i><span style="font-size: x-small;">Figure. The effects of HDAC2 siRNA Infusion into the CeA of P rats on voluntary ethanol consumption as measured by the two-bottle free choice paradigm. Monitoring the voluntary ethanol consumption of alcohol-preferring (P) rats via the two bottle free choice paradigm following infusion of vehicle or histone deacetylase isoform 2 (HDAC2) siRNA into the central amygdala (CeA) demonstrates that high HDAC2 levels may mediate the high alcohol drinking behaviors of P rats. P rats were given access to water and 7% ethanol followed by water and 9% ethanol. On the sixth day of ethanol access P rats received infusion of vehicle or HDAC2 siRNA and consumption of water and 9% ethanol were monitored for sevnfusion. Total fluid intake did not significantly differ between the groups. Values are represented as the mean ± SEM of the ethanol consumption (g / kg / day) and total fluid intake (mL) plotted daily for n=6 rats per treatment group. *Significantly different between the groups.</span></i><br />
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This data suggest reduction of HDAC2 levels in the CeA leads to reduced DSD associated with a reduction in anxiety-like behaviors and alcohol preference in P rats and could prove to have therapeutic value.Neuromicshttp://www.blogger.com/profile/08144200376477478451noreply@blogger.com0tag:blogger.com,1999:blog-4594982080375426081.post-7044135224869519462014-08-28T11:08:00.000-07:002014-08-28T11:08:36.198-07:00miRNA, Inflammation and Coronary Heart Diseasei-Fect<sup>TM</sup> Delivers miRNA for the Study of Cardiovascular Pathogenesis <br />
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We have posted over <a href="http://www.neuromics.com/ittrium/visit/A1x66x1y1x85b0x1x9dy1xe3x1y1xd0ex1y1xd26x1" target="_blank">35 publications</a> that reference use of our <a href="http://www.neuromics.com/ittrium/visit/A1x66x1y1x85b1x1x9cy1x622fx1x96y1x351dx1x82" target="_blank">i-Fect Transfection Kit</a> to deliver siRNA, miRNA and shRNA <i>in vitro</i> and <i>in vivo</i>. Results documented in these publications prove that this kit is both non-toxic and delivers ultra-high transfection efficiency.<br />
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Here i-Fect is used to silence miR-21 microRNAs. This miRNA stimulates pro-inflammatory pathways that are at the root of Coronary Heart Disease: <a href="http://www.cmj.org/ch/reader/view_abstract.aspx?file_no=20140780&flag=1" target="_blank">Guo Weizao, Liu Huichen, Li Lin, Yang Man and Du Aihua. Regulation of lovastatin on a key inflammation-related microRNA in myocardial cells</a>. Chinese Medical Journal 2014;127(16):2977-2981:10.3760/cma.j.issn.0366-6999.20140780...... miRNA functional inhibition assay Anti-miR miRNA antagonist for miR-21 (Ambion/Life Technologies, Grand Island, NY, USA) was transfected into H9c2(2-1) cells using iFect transfection kit (Neuromics, Edina, MN, USA) according to the manufacturer's manual...<br />
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<b>Results:</b>Inhibition of miR-21 upregulates STAT-3 and exerts a critical role in the upregulation of cardioprotective and anti-apoptotic proteins.<br />
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<i>Fig: Inhibition of miR-21 attenuated the up-regulation of phosphorylation of STAT3 in H9c2(2-1) cells by lovastatin (LST) in lipopolysaccharide (LPS) treated cardiomyocytes. Combination of treatments are indicated under the image, the basic comparison was 1 vs 3. </i><br />
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<i></i>This study demonstrates the relationship between miR-21 and the STAT3 pathway in Coronary Heart Disease. Delivering inhibitory miRNA into cardiomyocytes was key to establishing this relationship. Further study could enable discovery of STAT3 related targets for CV protective drug. In the spirit of helping researchers find the best solutions and protocols for Gene Expression Analysis Studies, we will continue to post new findings.Neuromicshttp://www.blogger.com/profile/08144200376477478451noreply@blogger.com0tag:blogger.com,1999:blog-4594982080375426081.post-38471572908268588592014-07-23T11:40:00.001-07:002014-07-23T11:43:10.194-07:00Neuromics' Transfection Kits-Genes Studied<strong><em>Delivering siRNA, miRNA, Plasmids and Viral Vectors for Gene Expression Analysis</em></strong>.<br />
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I have shared the many genes researchers have studied using our <a href="http://β-arrestin/" target="_blank">Transfection Kits</a>. 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!<br />
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<a href="http://link.springer.com/article/10.1007/s12031-014-0379-3" target="_blank">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</a>. Journal of Molecular Neuroscience. July 2014...Ten micrograms of siRNA1- GPNMB dissolved in 30 μl <a href="http://www.neuromics.com/ittrium/visit/A1x66x1y1x85b1x1x9cy1x622fx1x96y1x351dx1x82" target="_blank">i-Fect transfection reagent</a> (Neuromics, Edina, MN, USA) was administered intrathecally once daily for 7 days, starting from 1 day before CCI surgery... <br />
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<strong>Abstract:</strong> 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. <br />
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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. Neuromicshttp://www.blogger.com/profile/08144200376477478451noreply@blogger.com0tag:blogger.com,1999:blog-4594982080375426081.post-23786727286964762292014-06-25T12:05:00.001-07:002014-06-25T12:05:32.482-07:00Gene Expression Analysis For Neuroscientists<b><i>In Vitro</i> and <i>In Vivo</i> Studies</b><br />
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<a href="http://www.neuromics.com/" target="_blank">Neuromics</a> has a strong line up of <a href="http://www.neuromics.com/transfection-kits-and-reagents" target="_blank">Transfection Tools</a> designed specifically for Neuroscientists. Neurons, Glia and Astrocytes are notoriously hard to transfect. We are proud of our track record.<br />
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This is an excellent study for Neuroscience Researchers interested in using best methods:<a href="http://emboj.embopress.org/content/embojnl/31/15/3239/DC1/embed/inline-supplementary-material-1.pdf?download=true" target="_blank"> http://emboj.embopress.org/content/embojnl/31/15/3239/DC1/embed/inline-supplementary-material-1.pdf?download=true. </a> Here researchers delivered 14-3-3 siRNA sub units + <a href="http://www.neuromics.com/ittrium/visit/A1x66x1y1x85b1x1x9cy1x622fx1x96y1x351dx1x82" target="_blank">i-Fect </a>intrathecally. Here's specific knockdown results:<br />
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<span style="font-size: x-small;"><i>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).</i></span><br />
<span style="font-size: x-small;"><i><br /></i></span>
Our customers have successfully studied many genes with our tools. Here's a sampling: <span style="background-color: #fff2cc;"><span style="color: #222222;">ABCA, ASIC, β-arrestin, CAV, CX3CR1, DOR, ELOVL4, IKBKAP, K+-ATPase, K<sub>V</sub>1.1, K<sub>V</sub>9.1 ,The β3 subunit of the Na+,K+-ATPase, NTS1, NAV</span><sub>1.8</sub>, NTS1, NOV, Raf-1, RANK, SNSR1, hTert, NOV, Survivin, TLR4, Troy and TRPV1</span><span style="background-color: white;">. Here're the related <a href="http://www.neuromics.com/ittrium/visit/A1x66x1y1x85b0x1x9dy1xe3x1y1xd0ex1y1xd26x1" target="_blank">publications</a>.</span>Neuromicshttp://www.blogger.com/profile/08144200376477478451noreply@blogger.com0tag:blogger.com,1999:blog-4594982080375426081.post-26273320024426821452014-03-11T06:36:00.000-07:002014-03-11T06:36:36.077-07:00i-Fect, BDNF and Irritable Bowel Syndrome (IBS)<b><i>Gene Expression Analysis Determines BDNF's Role in IBS</i></b><br />
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In this study, Researchers used <a href="http://www.neuromics.com/ittrium/visit/A1x66x1y1x85b1x1x9cy1x622fx1x96y1x351dx1x82" target="_blank">Neuromics' i-Fect<sup>TM </sup> Transfection Ki</a>t to deliver BDNF to determine effect on visceral
hypersensitivity (VHS): <a href="http://onlinelibrary.wiley.com/doi/10.1111/nmo.12326/abstract" target="_blank">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</a>. 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
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<span style="font-size: x-small;"><i><b>Intrathecal treatment with brain-derived neurotrophic factor (BDNF) antagonists reduced VMR to colorectal distension (CRD) in female chronic prenatal stress (CPS)+HeICS rats</b>. (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 </i></span><i style="font-size: small;">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).</i></div>
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<b>Conclusion</b>: 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.Neuromicshttp://www.blogger.com/profile/08144200376477478451noreply@blogger.com0tag:blogger.com,1999:blog-4594982080375426081.post-78929678520761505642013-11-06T15:28:00.000-08:002013-11-06T15:28:09.925-08:00Using i-Fect to Knock Down GLT-1 Gene in vivo<b><i>Pain Researchers continue to use our <a href="http://www.neuromics.com/ittrium/visit/A1x66x1y1x85b1x1x9cy1x622fx1x96y1x351dx1x82" target="_blank">i-Fect<sup>TM</sup> Transfection Kit</a> for Gene Expression Analysis</i></b><br />
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<a href="http://placeholder/">Valproate Prevents Dysregulation of Spinal Glutamate and Reduces the Development of Hypersensitivity in Rats After Peripheral Nerve Injury</a>. The Journal of Pain, Volume 14, Issue 11, November 2013, Pages 1485–1491 <a href="http://dx.doi.org/10.1016/j.jpain.2013.07.007">http://dx.doi.org/10.1016/j.jpain.2013.07.007</a>.<br />
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Researchers use the kit to deliver Glutamate Receptor 1 siRNA <i>in vivo</i>.<br />
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.Neuromicshttp://www.blogger.com/profile/08144200376477478451noreply@blogger.com0tag:blogger.com,1999:blog-4594982080375426081.post-61389607513003460212013-07-29T13:12:00.000-07:002013-07-29T13:14:13.122-07:00i-Fect siRNA Transfection Successes<b><i>Meeting your Gene Expression Analysis Requirements.</i></b><br />
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I have posted many success stories resulting from use of <a href="http://www.neuromics.com/ittrium/visit/A1x66x1y1x85b1x1x9cy1x622fx1x96y1x351dx1x82" target="_blank">Neuromics' Neuromics' i-Fect<sup>TM</sup> siRNA Transfection Kit</a>. It has been 3 months since my last posting so here I would like to post some recent highlights.
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Here I feature customer publication demonstrating:<br />
<ol>
<li>Down regulation of<span style="background-color: yellow;"> Neuroligin 2</span>-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.</li>
<li>Role of <span style="color: yellow;">I</span><span style="background-color: yellow;">GFPB5</span> for survival of neurons in post-natal development.</li>
<li><span style="background-color: yellow;">HDAC</span> role in Anxiety and Alcoholsim</li>
</ol>
Here're the publications:<br />
<span style="color: blue;"><a href="http://www.sciencedirect.com/science/article/pii/S030439591300403X" title="i-Fect siRNA Transfection Kit Publication"><span style="color: blue;">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</span></a>,</span> PAIN, Available online 25 July 2013, ISSN 0304-3959, http://dx.doi.org/10.1016/j.pain.2013.07.035.<br />
(<a href="http://www.sciencedirect.com/science/article/pii/S030439591300403X"><span style="color: blue;">http://www.sciencedirect.com/science/article/pii/S030439591300403X</span></a>)...<span style="background-color: white; color: black;">The siRNAs were solubilized in 10 µl of i-Fect reagent (</span><strong style="background-color: white;">Neuromics</strong><span style="background-color: white; color: black;">, Edina, Minnesota, USA) following </span><strong style="background-color: white;">Neuromics </strong><span style="background-color: white; color: black;">instructions and published protocol, and applied intrathecally using a Hamilton syringe...</span><br />
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<a href="http://www.nature.com/neuro/journal/v16/n5/full/nn.3358.html" title="i-Fect Transfection Kit-Customer Publication">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</a>. Nature Neuroscience 16, 543–551 (2013) doi:10.1038/nn.3358.<br />
...vehicle (PBS) was delivered intraventricularly through the cisterna magna with a glass pipette while P3 mice were cold anesthetized. <em>Igf1</em> siRNA (stealth siRNA, Invitrogen) or <em>Igfbp5</em> siRNA with i-Fect reagent (Neuromics) was delivered intraventricularly through the cisterna magna at P3 twice with a 12-h interval...<br />
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<a href="http://www.biologicalpsychiatryjournal.com/article/S0006-3223(13)00052-8/abstract" mce_href="http://www.biologicalpsychiatryjournal.com/article/S0006-3223(13)00052-8/abstract" title="i-Fect Customer Publication. Delivering siRNA to the Brain by Micro-injection">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</a>. Biological Psychiatry.
doi.org/10.1016/j.biopsych.2013.01.012<br />
...The siRNAs were dissolved in iFect solution (Neuromics, Edina)...<br />
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I will keep updating you as more pubs and data becomes available.Neuromicshttp://www.blogger.com/profile/08144200376477478451noreply@blogger.com0tag:blogger.com,1999:blog-4594982080375426081.post-44133532059618928242013-04-25T11:57:00.001-07:002013-04-25T12:00:30.459-07:00i-Fect Delivers IGF1 siRNA to the Mouse Brain<strong><em>Intraventricular Injections Used to Study Neuronal Survival During Post Natal Development</em></strong><br />
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Neuromics'<a href="http://www.neuromics.com/ittrium/visit/A1x66x1y1x85b1x1x9cy1x622fx1x96y1x351dx1x82y1xf75x1x7f" target="_blank"> i-Fect<sup>TM</sup> Transfection Kit</a> continue to be successfully used to deliver siRNA, shRNA and miRNA to cell cultures and the CNS <em>in vivo </em>(via intrathecal, epidural and intraventricular injection<span style="background-color: white;">). </span><span style="background-color: yellow;">Genes studied include: ABCA, ASIC, β-arrestin, CAV<sub>1.2</sub>, IGF1, CX3CR1, DOR, ELOVL4, IKBKAP, K+-ATPase, K<sub>V</sub>1.1, K<sub>V</sub>9.1 ,The β3 subunit of the Na+,K+-ATPase, NTS1, NAV<sub>1.8</sub>, NTS1, NOV, Raf-1, RANK,SNSR1, hTertTRPV1 NOV, Survivin, TLR4, Troy and TRPV1. </span><span style="background-color: white;"><a href="http://www.neuromics.com/ittrium/visit/A1x66x1y1x85b0x1x9dy1xe3x1y1xd0ex1y1xd26x1" target="_blank"><strong>Related Publications.</strong></a></span><br />
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<span style="background-color: white;">It is always an honor for one our products to be referenced in one of the <em>Nature</em> publications. Here researchers use i-Fect to study the impact of microglial derived IGF1 silencing on Neuronal Survival: <a href="http://www.nature.com/neuro/journal/v16/n5/full/nn.3358.html" target="_blank">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.</a> Nature Neuroscience 16, 543–551 (2013) doi:10.1038/nn.3358. ...<em>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</em>... </span><br />
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<span style="font-size: x-small;">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.</span><br />
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I will continue to post new developments and successes.Neuromicshttp://www.blogger.com/profile/08144200376477478451noreply@blogger.com0