Neuromics' Customers have published their success using i-FectTM for gene expression studies Genes studied include: DOR, hTERT, The β3 subunit of the Na+,K+-ATPase, rSNSR1, NTS1. NAV1.8 and more.
We are now pleased to add knockdown L Calcium Channel Subtypes to study differential effects of neuropathic pain.
In this study researchers showed specific knockdown of CaV1.2 in the spinal dorsal horn reversed the neuropathy-associated mechanical hypersensitivity and the hyperexcitability and increased responsiveness of dorsal horn neurons. Intrathecal application of anti-CaV1.2 siRNAs confirmed the preceding results.
Here's a link to the related pub: Pascal Fossat, Eric Dobremez, Rabia Bouali-Benazzouz, Alexandre Favereaux, Sandrine S. Bertrand, Kalle Kilk, Claire Léger, Jean-René Cazalets, Ülo Langel, Marc Landry and Frédéric Nagy. Knockdown of L Calcium Channel Subtypes: Differential Effects in Neuropathic Pain. The Journal of Neuroscience, January 20, 2010, 30(3):1073-1085; doi:10.1523/JNEUROSCI.3145-09.2010
We used siRNA targeting several splice variants of CaV1.2 ("Silencer Select Pre-designed and Validated siRNA", Ambion). They consisted of a pool of two 21 nt duplex. siRNAs were selected to target two distinct CaV1.2 mRNA regions to enhance silencing. The antisense sequences were as follows: UCUAUUGUCAUAUCGCAGG and UAUCCGAACAGGUAUAGAG.
In contrast to PNA, these siRNAs targeted the 5'-coding region. Mismatch siRNA was a nontargeting 21 nt duplex designed as a negative control. The siRNAs (2 µg) were solubilized in 10 µl of reagent i-Fect (Neuromics) following Neuromics instructions and published protocol (Luo et al., 2005), and applied intrathecally according to the same protocol as for the PNA.
Thursday, February 18, 2010
Thursday, January 7, 2010
Delivering siRNA in Mice for Studying Opioid-Induced Hyperalgesia
Researchers have successfully delivered siRNA in-vitro and in-vivo using Neuromics' i-Fect ™ siRNA Transfection Reagent. Gene expression studies include: DOR, hTERT, The β3 subunit of the Na+,K+-ATPase, rSNSR1, NTS1. NAV1.8 and more.
Here's a link to all transfection publications: Transfection Kit Pubs
We are pleased to present yet another study and related publication. This includes one of the first successful delivery of siRNA in mice using i-Fect ™ :
Yan Chen, Cheng Yang, and Zaijie Jim Wang. Ca2+/Calmodulin-Dependent Protein Kinase II Is Required for the Initiation and Maintenance of Opioid-Induced Hyperalgesia. The Journal of Neuroscience, January 6, 2010, 30(1):38-46; doi:10.1523/JNEUROSCI.4346-09.2010.
...KN93 and KN92 were administered intrathecally by percutaneous puncture through the L5-L6 intervertebral space, as described previously (Hylden and Wilcox, 1980; Chen et al., 2009). A lateral tail flick was considered as success of the intrathecal injection. To inhibit CaMKII, CaMKII was targeted by small interfering RNA (siRNA). Four days after morphine pellet implantation, mice were treated with CaMKII siRNA (5'-CACCACCAUUGAGGACGAAdTdT-3', 3'-dTdTGUGGUGGUAACUCCUGCUU-5') (Zayzafoon et al., 2005) or Stealth RNAi negative control (Invitrogen) (2 µg, i.t., twice per day for 3 consecutive days). These oligos were mixed with the transfection reagent i-Fect (Neuromics), in a ratio of 1:5 (w/v) (Luo et al., 2005). Mechanical and thermal sensitivity tests were performed daily...
Here's a link to all transfection publications: Transfection Kit Pubs
We are pleased to present yet another study and related publication. This includes one of the first successful delivery of siRNA in mice using i-Fect ™ :
Yan Chen, Cheng Yang, and Zaijie Jim Wang. Ca2+/Calmodulin-Dependent Protein Kinase II Is Required for the Initiation and Maintenance of Opioid-Induced Hyperalgesia. The Journal of Neuroscience, January 6, 2010, 30(1):38-46; doi:10.1523/JNEUROSCI.4346-09.2010.
...KN93 and KN92 were administered intrathecally by percutaneous puncture through the L5-L6 intervertebral space, as described previously (Hylden and Wilcox, 1980; Chen et al., 2009). A lateral tail flick was considered as success of the intrathecal injection. To inhibit CaMKII, CaMKII was targeted by small interfering RNA (siRNA). Four days after morphine pellet implantation, mice were treated with CaMKII siRNA (5'-CACCACCAUUGAGGACGAAdTdT-3', 3'-dTdTGUGGUGGUAACUCCUGCUU-5') (Zayzafoon et al., 2005) or Stealth RNAi negative control (Invitrogen) (2 µg, i.t., twice per day for 3 consecutive days). These oligos were mixed with the transfection reagent i-Fect (Neuromics), in a ratio of 1:5 (w/v) (Luo et al., 2005). Mechanical and thermal sensitivity tests were performed daily...
Tuesday, December 1, 2009
Using i-Fect for treatment of Glioblastomas
Dr. Swapan K. Ray, Department of Pathology, Microbiology, and Immunology, University of South Carolina School of Medicine and his team should positive results in reducing growth of Glioblastomas by knocking down hTERT expression using Neuromics' i-Fect ™ siRNA Transfection Kit. Here's the related pub:
Joseph George, Naren L. Banik, Swapan K. Ray. Combination of hTERT Knockdown and IFN-γ Treatment Inhibited Angiogenesis and Tumor Progression in Glioblastoma. Clin Cancer Res 2009;15(23):7186–95
...with i-Fect transfection reagent (Neuromics) to obtain 5 μg DNA/10 μL of injection volume...
Results: In vitro and in vivo angiogenesis assays showed inhibition of capillary-like network formation of microvascular endothelial cells and neovascularization under dorsal skin of nude mice, respectively. We observed inhibition of intracerebral tumorigenesis and s.c. solid tumor formation in nude mice after treatment with combination of hTERT siRNA and IFN-γ. Western blotting of solid tumor samples showed significant downregulation of the molecules that regulate cell invasion, angiogenesis, and tumor progression.
Conclusions: Our study showed that the combination of hTERT siRNA and IFN-γ effectively inhibited angiogenesis and tumor progression through the downregulation of molecules involved in these processes. Therefore, the combination of hTERT siRNA and IFN-γ is a promising therapeutic strategy for controlling the growth of human glioblastoma.
Joseph George, Naren L. Banik, Swapan K. Ray. Combination of hTERT Knockdown and IFN-γ Treatment Inhibited Angiogenesis and Tumor Progression in Glioblastoma. Clin Cancer Res 2009;15(23):7186–95
...with i-Fect transfection reagent (Neuromics) to obtain 5 μg DNA/10 μL of injection volume...
Results: In vitro and in vivo angiogenesis assays showed inhibition of capillary-like network formation of microvascular endothelial cells and neovascularization under dorsal skin of nude mice, respectively. We observed inhibition of intracerebral tumorigenesis and s.c. solid tumor formation in nude mice after treatment with combination of hTERT siRNA and IFN-γ. Western blotting of solid tumor samples showed significant downregulation of the molecules that regulate cell invasion, angiogenesis, and tumor progression.
Conclusions: Our study showed that the combination of hTERT siRNA and IFN-γ effectively inhibited angiogenesis and tumor progression through the downregulation of molecules involved in these processes. Therefore, the combination of hTERT siRNA and IFN-γ is a promising therapeutic strategy for controlling the growth of human glioblastoma.
Tuesday, June 23, 2009
Delivering 27mer DsiRNAs to Mice DRGs
I have been a proponent of using 27mer DsiRNAs (Dicer Substrate Small Interfering RNAs) with our i-Fect kits to deliver siRNA to the CNS for gene expression analysis. The potency of this platform was highlighted in my profile of Dr. Mark Behlke.
It was further confirmed by in Studies conducted by Dr. Philippe Serrat and his team at University of Sherbrooke.
Louis Doré-Savard, Geneviève Roussy, Marc-André Dansereau, Michael A Collingwood, Kim A Lennox, Scott D Rose, Nicolas Beaudet, Mark A Behlke and Philippe Sarret. Central Delivery of Dicer-substrate siRNA: A Direct Application for Pain Research. Molecular Therapy (2008); Jul;16(7):1331-9. Epub 2008 Jun 3 doi:10.1038/mt.2008.98.
Using ultra low dose of DsiRNAs complexed with Neuromics’ i-Fect , they were able to successfully reduce NTS2 gene expression by up to 86% in rat lumbar Dorsal Root Ganglia after only two intrathecal injections. This was confirmed by Western Blot and qPCR analysis.
We now have further confirmation of the capabilities of this delivery platform in a just released publication by Dr. Jeffrey Mogil and team:
Michael L. LaCroix-Fralish, Gary Mo, Shad B. Smith, Susana G. Sotocinal, Jennifer Ritchie, Jean-Sebastien Austin, Kara Melmed, Ara Schorscher-Petcu, Audrey C. Laferriere, Tae Hoon Lee, Dmitry Romanovsky, Guochun Liao, Mark A. Behlke, David J. Clark, Gary Peltz, Philippe Séguéla, Maxim Dobretsov and Jeffrey S. Mogil. The β3 subunit of the Na+,K+-ATPase mediates variable nociceptive sensitivity in the formalin test. doi:10.1016/j.pain.2009.04.028.
IT Delivery of siRNA in vivo supplement
It was further confirmed by in Studies conducted by Dr. Philippe Serrat and his team at University of Sherbrooke.
Louis Doré-Savard, Geneviève Roussy, Marc-André Dansereau, Michael A Collingwood, Kim A Lennox, Scott D Rose, Nicolas Beaudet, Mark A Behlke and Philippe Sarret. Central Delivery of Dicer-substrate siRNA: A Direct Application for Pain Research. Molecular Therapy (2008); Jul;16(7):1331-9. Epub 2008 Jun 3 doi:10.1038/mt.2008.98.
Using ultra low dose of DsiRNAs complexed with Neuromics’ i-Fect , they were able to successfully reduce NTS2 gene expression by up to 86% in rat lumbar Dorsal Root Ganglia after only two intrathecal injections. This was confirmed by Western Blot and qPCR analysis.
We now have further confirmation of the capabilities of this delivery platform in a just released publication by Dr. Jeffrey Mogil and team:
Michael L. LaCroix-Fralish, Gary Mo, Shad B. Smith, Susana G. Sotocinal, Jennifer Ritchie, Jean-Sebastien Austin, Kara Melmed, Ara Schorscher-Petcu, Audrey C. Laferriere, Tae Hoon Lee, Dmitry Romanovsky, Guochun Liao, Mark A. Behlke, David J. Clark, Gary Peltz, Philippe Séguéla, Maxim Dobretsov and Jeffrey S. Mogil. The β3 subunit of the Na+,K+-ATPase mediates variable nociceptive sensitivity in the formalin test. doi:10.1016/j.pain.2009.04.028.
IT Delivery of siRNA in vivo supplement
Monday, April 20, 2009
Knockdown of rSNSR1 in vivo
The parade of success with use our i-FectTM in vivo grows. Here's the most recent study:
Christian Ndong, Amynah Pradhan, Carole Puma, Jean-Pierre Morello, Cyrla Hoffert, Thierry Groblewski , Dajan O’Donnell, Jennifer M.A. Laird. Role of rat sensory neuron-specific receptor (rSNSR1) in inflammatory pain: Contribution of TRPV1 to SNSR signaling in the pain pathway. PAIN 143 (2009) 130–137.
...For experiments in which siRNA was delivered by bolus injections, 10 ul of siRNA or vehicle was injected directly into the intrathecal catheter once daily for 4 days. In this case, siRNAs were prepared immediately prior to administration by mixing the RNA solution (200 uM in annealing buffer) with the transfection reagent i-FectTM (Neuromics) at a ratio of 1:4 (w:v) for a final siRNA/ lipid complex concentration of 2 ug/10 ul...
Related Data:
Images: in vivo characterization of knockdown produced by rSNSR1 siRNA. (A) A dose-dependent decrease in rSNSR1 mRNA levels measured in lumbar L3/L4/L5 DRGs was
observed when rSNSR1 siRNA (n = 7–14/group) or MM siRNA (n = 6/group) was delivered by four daily bolus injections. *p < 0.05; **p < 0.01; ***p < 0.001 as determined by oneway analysis of variance followed by sequential testing. (B) rSNSR1 immunoreactivity in dorsal horn of the spinal cord was visibly reduced in rSNSR1 siRNA-treated animals (5 lg/day, left panel). Immunoreactivity with neuron-specific isolectin B4 (IB4; right panel) did not change between treatment groups, showing the integrity of each dorsal horn analyzed (n = 6/group). (C) A semi-quantitative score of rSNSR1 immunoreactivity showed that siRNA treatment greatly decreased rSNSR1 protein levels compared to MM and control groups. A blinded observer scored 9–12 individual sections taken from a 1 cm segment of the spinal cord.
Christian Ndong, Amynah Pradhan, Carole Puma, Jean-Pierre Morello, Cyrla Hoffert, Thierry Groblewski , Dajan O’Donnell, Jennifer M.A. Laird. Role of rat sensory neuron-specific receptor (rSNSR1) in inflammatory pain: Contribution of TRPV1 to SNSR signaling in the pain pathway. PAIN 143 (2009) 130–137.
...For experiments in which siRNA was delivered by bolus injections, 10 ul of siRNA or vehicle was injected directly into the intrathecal catheter once daily for 4 days. In this case, siRNAs were prepared immediately prior to administration by mixing the RNA solution (200 uM in annealing buffer) with the transfection reagent i-FectTM (Neuromics) at a ratio of 1:4 (w:v) for a final siRNA/ lipid complex concentration of 2 ug/10 ul...
Related Data:
Images: in vivo characterization of knockdown produced by rSNSR1 siRNA. (A) A dose-dependent decrease in rSNSR1 mRNA levels measured in lumbar L3/L4/L5 DRGs was
observed when rSNSR1 siRNA (n = 7–14/group) or MM siRNA (n = 6/group) was delivered by four daily bolus injections. *p < 0.05; **p < 0.01; ***p < 0.001 as determined by oneway analysis of variance followed by sequential testing. (B) rSNSR1 immunoreactivity in dorsal horn of the spinal cord was visibly reduced in rSNSR1 siRNA-treated animals (5 lg/day, left panel). Immunoreactivity with neuron-specific isolectin B4 (IB4; right panel) did not change between treatment groups, showing the integrity of each dorsal horn analyzed (n = 6/group). (C) A semi-quantitative score of rSNSR1 immunoreactivity showed that siRNA treatment greatly decreased rSNSR1 protein levels compared to MM and control groups. A blinded observer scored 9–12 individual sections taken from a 1 cm segment of the spinal cord.
Monday, April 6, 2009
Did Your RNAi Experiment Work?
This is a good methods publication on RNAi transfection: Reliably Validating RNA Interference with qRT-PCR.
Bill Wang, Song Tian, Qiong Zhou, and Xiao Zeng. SA Biosciences.
Bill Wang, Song Tian, Qiong Zhou, and Xiao Zeng. SA Biosciences.
Tuesday, December 16, 2008
Intrathecal Delivery of siRNA
We wanted to present yet another publication referencing successful delivery of siRNA using i-FectTM:
Suneeta Tumati, Tally Largent Milnes, Henry I. Yamamura, Todd W. Vanderah, William R. Roeske and Eva V. Varga. Intrathecal Raf-1-selective siRNA attenuates sustained morphine-mediated thermal hyperalgesia. doi:10.1016/j.ejphar.2008.10.033
...siRNAs stock solutions (100 μM) were prepared in double distilled RNAse free water and stored in aliquots at −80 °C. For intrathecal treatment, aliquots of the stock solution (2 μg of the appropriate siRNA) were mixed (1:5 v/v)with i-Fect transfection reagent (Neuromics, Edina, MN). After recovery from the surgery (5–7 days), the animals received intrathecal injections (2 ug siRNA/10 ul/rat) of either a lipid encapsulated Raf-1-selective siRNA mixture (Smart pool siRNA, Dharmacon Inc; Chicago, IL, Cat # L-087699-00) (Raf-1 siRNA groups) or i-Fect encapsulated non-targeting dsRNA (Dharmacon, #D-001810-01-20) (control mismatch siRNA groups) or the transfection lipid alone, once daily, for 3 days, as described earlier (Gardell et al., 2002). Intrathecal injections of the siRNAs or the transfection agent alone did not cause any sign of behavioral toxicity. Western blots, using a Raf-1-selective antibody, indicated that intrathecal treatment with the Raf-1-selective siRNA mixture for 3 days significantly reduced Raf-1 protein levels in the dorsal root ganglion and in the dorsal horn of the spinal cord...
Suneeta Tumati, Tally Largent Milnes, Henry I. Yamamura, Todd W. Vanderah, William R. Roeske and Eva V. Varga. Intrathecal Raf-1-selective siRNA attenuates sustained morphine-mediated thermal hyperalgesia. doi:10.1016/j.ejphar.2008.10.033
...siRNAs stock solutions (100 μM) were prepared in double distilled RNAse free water and stored in aliquots at −80 °C. For intrathecal treatment, aliquots of the stock solution (2 μg of the appropriate siRNA) were mixed (1:5 v/v)with i-Fect transfection reagent (Neuromics, Edina, MN). After recovery from the surgery (5–7 days), the animals received intrathecal injections (2 ug siRNA/10 ul/rat) of either a lipid encapsulated Raf-1-selective siRNA mixture (Smart pool siRNA, Dharmacon Inc; Chicago, IL, Cat # L-087699-00) (Raf-1 siRNA groups) or i-Fect encapsulated non-targeting dsRNA (Dharmacon, #D-001810-01-20) (control mismatch siRNA groups) or the transfection lipid alone, once daily, for 3 days, as described earlier (Gardell et al., 2002). Intrathecal injections of the siRNAs or the transfection agent alone did not cause any sign of behavioral toxicity. Western blots, using a Raf-1-selective antibody, indicated that intrathecal treatment with the Raf-1-selective siRNA mixture for 3 days significantly reduced Raf-1 protein levels in the dorsal root ganglion and in the dorsal horn of the spinal cord...
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