i-FectTMTranfection Kits***Delivery of siRNA, shRNA and miRNA in vivo & in vitro*

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. 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...

M. Leinders, b, N. Üçeyler, R.A. Pritchard, C. Sommer, L.S. Sorkin. Increased miR-132-3p expression is associated with chronic neuropathic pain. 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...

C.R. Lin, J.K. Cheng,C.H. Wu,K.H. Chen,C.K. Liu. Epigenetic suppression of potassium-chloride co-transporter 2 expression in inflammatory pain induced by complete Freund's adjuvant (CFA). European Journal of Pain. 10 August 2016. DOI: 10.1002/ejp.925

... Group NS control: normal saline injection (n = 5 per time point, total = 25); Group CFA: CFA-injected rats (n = 5 per time point, total = 25); Group SiCont: normal rats receiving intrathecal 2 μg/10 μL control siRNA+ transfection reagent (i-Fect, Neuromics Antibodies, Northfield, MN ...

Melissa T. Manners, Adam Ertel, Yuzhen Tian and Seena K. Ajit. Genome-wide redistribution of MeCP2 in dorsal root ganglia after peripheral nerve injury. 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 [27]. 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 iFECT 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...

Xiaobo Yanga, Heling Chua,Yuping Tanga,& Qiang Donga. The role of connexin43 in hemorrhagic transformation after thrombolysis in vivo and in vitro. Neuroscience. Available online 30 April 2016. doi:10.1016/j.neuroscience.2016.04.040

...The siRNA was prepared immediately prior to administration by mixing the RNA solution (1 mg/ml in annealing buffer) with the i-Fect transfection reagent (1/3, v/v; Neuromics, Edina, MN, USA) to a final siRNA/lipid complex concentration of 0.25 mg/ml. ...

Figueroa Johnny D., Serrano-Illan Miguel, Licero Jenniffer, Cordero Kathia, Miranda Jorge D., and De Leon Marino.Fatty Acid Binding Protein 5 Modulates Docosahexaenoic Acid-Induced Recovery in Rats Undergoing Spinal Cord Injury. Journal of Neurotrauma. March 2016, ahead of print. doi:10.1089/neu.2015.4186.

... catheters attached to an osmotic mini-pump. To facilitate delivery and uptake, siRNA aliquots were mixed (1:5, v/v) with the cationic lipid based transfection reagent i-FectTM (Neuromics, Edina, MN). Injections A group of animals were ...

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. 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...

Liuming Jiang, Qun Wu , Tao Yang. Silencing of Id2 Alleviates Chronic Neuropathic Pain Following Chronic Constriction Injury. Journal of Molecular Neuroscience\pp 1-7.First online: 15 January 2016

...Thereafter, the catheter was fixed and the incision was sealed. Then, 10 μg shRNA-Id2 dissolved in 30 μl i-Fect transfection reagent (Neuromics, Edina, MN, USA) was administered intrathecally once daily for 7 days...

Johnny D Figueroa, Miguel S Illan, Jenniffer Licero, Kathia Cordero, Jorge David Miranda, and Marino De Leon. Fatty acid binding protein 5 (FABP5) modulates docosahexaenoic acid (DHA)-induced recovery in rats undergoing spinal cord injury. Journal of Neurotrauma. December 2015, ahead of print. doi:10.1089/neu.2015.4186.

... To facilitate delivery and uptake, siRNA aliquots were mixed (1:5v/v) with the cationic lipid based transfection reagent i-Fect™ (Neuromics, Edina, MN)...

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. Nature Neuroscience (2015) doi:10.1038/nn.4165.

I-Fect Delivers euchromatic histone-lysine N-methyltransferase-2 (G9a) in vitro and in vivo.

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. 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

...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, i-Fect™ (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)...

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. 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 ...

Y. Xiang, T. Liu, H. Yang, F. Gao, H. Xiang, A. Manyande, Y. Tian and X. Tian. NRG1-ErbB signalling promotes microglia activation contributing to incision-induced mechanical allodynia. Article first published online: 27 AUG 2014. DOI: 10.1002/ejp.590. © 2014 European Pain Federation - EFIC®

...The targeting or scramble control siRNA was mixed with transfection reagent i-Fect™ (Neuromics, Minneapolis, MN, USA) in a ratio of 1:5 (W : V) (Luo et al., 2005; Chen et al., 2010) at a final concentration of 0.4 μg/μL right prior to it injection (2 μg, twice a day in a 12-h interval)....

SACHIN MOONAT. The Role of Amygdaloid Chromatin and Synaptic Remodeling in Anxiety and Alcoholism. 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

Guo Weizao, Liu Huichen, Li Lin, Yang Man and Du Aihua. Regulation of lovastatin on a key inflammation-related microRNA in myocardial cells. 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. ...

Lili Hou, Yanfeng Zhang, Yong Yang, Kai Xiang, Qindong Tan, Qulian Guo. Intrathecal siRNA Against GPNMB Attenuates Nociception in a Rat Model of Neuropathic Pain. Journal of Molecular Neuroscience. July 2014.

...Ten micrograms of siRNA1- GPNMB dissolved in 30 μl i-Fect transfection reagent (Neuromics, Edina, MN, USA) was administered intrathecally once daily for 7 days, starting from 1 day before CCI surgery...

J. H. Winston1 Q. Li1, S. K. Sarna1. Chronic prenatal stress epigenetically modifies spinal cord BDNF expression to induce sex-specific visceral hypersensitivity in offspring. Article first published online: 4 MAR 2014. Neurogastroenterology & Motility. DOI: 10.1111/nmo.12326. The Journal of Pain, Volume 14, Issue 11, November 2013, Pages 1485–1491 http://dx.doi.org/10.1016/j.jpain.2013.07.007

...Thirty-two gage catheters were inserted through the atlanto-occipital membrane and extended caudal of the lumbar enlargement. The location of each catheter was confirmed following euthanasia. Rats received either BDNF antagonist trkB-Fc (R&D Systems, Minneapolis, MN, USA), 5 μg in 10 μL sterile saline or vehicle once/day during the 9-day stress period or BDNF siRNA or control siRNA (2 μg of the appropriate siRNA; 1:5 v/v with i-Fect transfection reagent Neuromics, Edina, MN, USA). Each rat received 2 μg siRNA/10 μL) on days 1, 3, 5 and 7 of the 9 day stress period...

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

... diluted with the transfection reagent (i-Fect; Neuromics, Edina, MN) to achieve a final concentration of .17 nmol/10 μL, and intrathecally injected for 5 days in normal or...

Tiphaine Dolique, Alexandre Favereaux, Olivier Roca-Lapirot, Virginie Roques, Claire Léger, Marc Landy, Frédéric Nagy, Unexpected association of the “inhibitory” Neuroligin 2 with excitatory PSD95 in neuropathic pain, PAIN, Available online 25 July 2013, ISSN 0304-3959, http://dx.doi.org/10.1016/j.pain.2013.07.035. (http://www.sciencedirect.com/science/article/pii/S030439591300403X)

...The siRNAs were solubilized in 10 µl of i-Fect reagent (Neuromics, Edina, Minnesota, USA) following Neuromics instructions and published protocol, and applied intrathecally using a Hamilton syringe...

Masaki Ueno,Yuki Fujita, Tatsuhide Tanaka, Yuka Nakamura, Junichi Kikuta, Masaru Ishii & Toshihide Yamashita. Layer V cortical neurons require microglial support for survival during postnatal development. Nature Neuroscience 16, 543–551 (2013) doi:10.1038/nn.3358.

...vehicle (PBS) was delivered intraventricularly through the cisterna magna with a glass pipette while P3 mice were cold anesthetized. Igf1 siRNA (stealth siRNA, Invitrogen) or Igfbp5 siRNA with i-Fect reagent (Neuromics) was delivered intraventricularly through the cisterna magna at P3 twice with a 12-h interval...

Sachin Moonat, Amul J. Sakharkar, Huaibo Zhang, Lei Tanga, Subhash C. Pandey. Aberrant Histone Deacetylase2–Mediated Histone Modifications and Synaptic Plasticity in the Amygdala Predisposes to Anxiety and Alcoholism. Biological Psychiatry. doi.org/10.1016/j.biopsych.2013.01.012

...The siRNAs were dissolved in iFect solution (Neuromics, Edina)...

John H. Winston, Sushil K. Sarna. Developmental Origins of Functional Dyspepsia-Like Gastric Hypersensitivity in Rats. Gastroenterology. Volume 144, Issue 3, March 2013, Pages 570–579.e3. dx.doi.org/10.1053/j.gastro.2012.11.001.

...intrathecal treatment, 2 μg of the appropriate siRNA was mixed (1:5 vol/vol) with i-Fect transfection reagent (Neuromics, Edina, MN); rats received 2 ug siRNA/10 uL/rat/injection...

Tsantoulas C, Zhu L, Shaifta Y, Grist J, Ward JP, Raouf R, Michael GJ, McMahon SB. Sensory neuron downregulation of the Kv9.1 potassium channel subunit mediates neuropathic pain following nerve injury. J Neurosci. 2012 Nov 28;32(48):17502-13. doi: 10.1523/JNEUROSCI.3561-12.2012.

..."I'd just like to notify you about a recent paper from my team which utilised iFect for in vivo transfection of dorsal root ganglia." Dr. Christoforos Tsantoulas, University of Cambridge...i-Fect-siRNA-mediated knock-down of Kv9.1 in naive rats led to neuropathic pain behaviors...

Masaki Ueno, Yasufumi Hayano1, Hiroshi Nakagawa and Toshihide Yamashita. Intraspinal rewiring of the corticospinal tract requires target-derived brain-derived neurotrophic factor and compensates lost function after brain injury. Brain (2012) doi: 10.1093/brain/aws053.

... motor cortex at 14 days after the injury, using i-Fect™ transfection reagents (Neuromics) according to the manufacture's instruction...

Christoforos Tsantoulas, Lan Zhu, Yasin Shaifta, John Grist, Jeremy P. T. Ward, Ramin Raouf, Gregory J. Michael, and Stephen B. McMahon. Sensory Neuron Downregulation of the Kv9.1 Potassium Channel Subunit Mediates Neuropathic Pain following Nerve Injury. The Journal of Neuroscience, 28 November 2012, 32(48): 17502-17513; doi: 10.1523/​JNEUROSCI.3561-12.2012.

...In vivo RNA interference: Anesthetized rats were subjected to a thoracic laminectomy and a silastic tube was inserted subdurally to lie just rostral to L3 DRG and externalized to deliver bolus injections (one injection per day for 4 consecutive days). Animals were allowed to recover for 5 d before treatment commenced. On the day of injection, siRNA was mixed with i-Fect (Neuromics) to a final concentration of 0.2 μg μl−1, according to published protocols (Luo et al., 2005). For each treatment, 10–20 μl of Kv9.1 siRNA or scrambled control mixture was injected, followed by a 10 μl saline flush. Twenty-four hours after the fourth injection animals were killed and L5 DRGs fresh dissected for qRT-PCR analysis. A separate set of animals were PFA perfused and DRGs retrieved for IHC. Passenger strand sequences for Kv9.1 and scrambled control siRNAs were cuuggaaucuguaggauca and gaggcctaatcgatatgtt, respectively (Dharmacon; “in vivo processing” option)...

Jing Chen, Xiaolu Zhang, Handojo Kusumo, Lucio G. Costa, c, Marina Guizzettia. Cholesterol efflux is differentially regulated in neurons and astrocytes: Implications for brain cholesterol homeostasis. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 1831 (2), p.263-275, Feb 2013. doi:10.1016/j.bbalip.2012.09.007

...transfection was carried out by adding to the cultures a solution containing 12 nM ABCG1 or ABCG4 SiRNA, i-Fect siRNA Transfection Reagent, and Opti-MEM I for 24 h. Exogenous cholesterol efflux was measured 48 h after the removal of the transfection reagents. The specific silencing of ABCG1 in astrocytes and neurons was confirmed by Western blot and by qRT-PCR; silencing of ABCG4 was confirmed only by qRT-PCR because no specific antibody to ABCG4 is available...

Sylvie Diochot, Anne Baron, Miguel Salinas, Dominique Douguet, Sabine Scarzello, Anne-Sophie Dabert-Gay, Delphine Debayle, Valérie Friend, Abdelkrim Alloui, Michel Lazdunski, Eric Lingueglia. Black mamba venom peptides target acid-sensing ion channels to abolish pain. Nature (2012)doi:10.1038/nature11494.

...Locally designed siRNAs targeting ASIC1 (si-ASIC1a/1b, GCCAAGAAGUUCAACAAAUdtdt), ASIC2 (si-ASIC2a/2b, UGAUCAAAGAGAAGCUAUUdtdt) and ASIC2a (si-ASIC2a, AGGCCAACUUCAAACACUAdtdt) have been validated in vitro in COS-7 cells transfected with myc-ASIC1a, ASIC1b, myc-ASIC2a or myc-ASIC2b, and the relevant siRNA or a control siRNA (si-CTR, GCUCACACUACGCAGAGAUdtdt) with TransIT-LT1 and transIT-TKO (Mirus), respectively. Cells were lysed 48 h after transfection and processed for western blot analysis to assess the amount of ASIC1a protein with the anti-Myc A14 antibody (1:500; Santa Cruz Biotechnology) or the anti-ASIC1 antibody (N271/44; 1:300; NeuroMab) and a monoclonal antibody against actin (AC-40; 1:1,000; Sigma) as a loading control. siRNAs were intrathecally injected into mice (2 µg per mouse at a ratio of 1:4 (w/v) with i-Fect (Neuromics)) twice a day for 3 days. After 3 days of treatment, the paw-flick latency was measured and the residual effect of mambalgin-1 (intrathecal or intraplantar, 34 µM) or the carrageenan (intraplantar, 2%)-induced hyperalgesia was tested. For validation of the in vivo effect of the siRNAs, lumbar DRGs or lumbar dorsal spinal cord were removed after the last siRNA injection for total RNA isolation (RNeasy kits, Qiagen) followed by cDNA synthesis (AMV First-Strand cDNA synthesis kit (Invitrogen) and High Capacity RNA-to-cDNA Kit, (Applied Biosystems)). The relative amounts of ASIC transcripts were evaluated by quantitative reverse-transcription PCR in a Light-Cycler 480 (Roche Products). Pre-designed and validated TaqMan assays for ASIC1 (ASIC1a and ASIC1b; Mm01305998_mH), ASIC1a (Mm01305996_m1), ASIC2 (ASIC2a and ASIC2b; Mm00475691_m1), ASIC3 (Mm00805460_m1) and 18S ribosomal RNA (Mm03928990_g1) were from Applied Biosystems. Each cDNA sample was run in triplicate and results were normalized against 18S and converted to fold induction relative to control siRNA treatment...

Shuangxin Liu, Wei Shi, Houqin Xiao, Xinling Liang, Chunyu Deng, Zhiming Ye, Ping Mei, Suxia Wang, Xiaoying Liu, Zhixin Shan, Yongzheng Liang, Bin Zhang, Wenjian Wang, Yanhui Liu, Lixia Xu, Yunfeng Xia, Jianchao Ma, Zhilian Li. Receptor Activator of NF-kappaB and Podocytes: Towards a Function of a Novel Receptor-Ligand Pair in the Survival Response of Podocyte Injury. PLoS ONE: Research Article, published 25 Jul 2012 10.1371/journal.pone.0041331

...RANK small interference RNA (siRNA) knockdown was performed by using transient transfection of pooled, functionally validated Cy3–labeled RANK siRNA (Invitrogen) [16]. Podocytes that were differentiated for 10 to 12 d were maintained at 10% FBS/RPMI as described above, and transfected using the RANK siRNA transfection reagent (Neuromics). For determination of the transfection efficiency, a Cy3–labeled RANK siRNA was analyzed by flow cytometry. Western blot analysis for RANK was performed with samples from cells 24 to 96 h after the transfection. Several concentrations of RANK siRNA (20, 40, and 60 nM) were tested to determine optimal knockdown conditions...

Jacobs VL, Liu Y, De Leo JA (2012) Propentofylline Targets TROY, a Novel Microglial Signaling Pathway. PLoS ONE 7(5): e37955. doi:10.1371/journal.pone.0037955.

Small interference RNA (siRNA) oligonucleotides specific for TROY (#1:s144862, #2:s144863, #3:s144864) were validated by and purchased from Invitrogen (Grand Island, NY). Transient transfection was carried out using iFect (Neuromics Edina, MN) as previously described. Briefly, microglia were plated at 3×105 cells/well in a 12-well plate. Once cells had adhered, they were transfected with 1 µg siRNA. Control samples were treated with empty vector siRNA (Sigma St Louis, MO) or iFect reagent alone. Cells were left in microglia media (10% fetal bovine serum (Hyclone Logan, UT), 1.1% GlutaMax (Invitrogen Carlsbad, CA), and 1% penicillin/streptomycin (100 U/ml penicillin, 100 µg/ml streptomycin, Mediatech, Manassas, VA)) at 37°C with 5% CO2 overnight and then used the following day for experiments...

Lara Kular, Cyril Rivat, Brigitte Lelongt, Claire Calmel, Maryvonne Laurent, Michel Pohl, Patrick Kitabgi, Stephane Melik-Parsadaniantz and Cecile Martinerie. NOV/CCN3 attenuates inflammatory pain through regulation of matrix metalloproteinases-2 and -9. Journal of Neuroinflammation 2012, 9:36. doi:10.1186/1742-2094-9-36. Published: 21 February 2012.

...For behavioral and protein analyses, rats were daily injected intrathecally under brie fisoflurane anesthesia with 2 μg of NOV siRNA (160 pmoles in i-Fect™  transfection reagent) or control siRNA on the day of CFA injection and then on the two following days (n = 8 rats/group). To test the NOV effect on pain intensity, three micrograms of commercial human NOV protein (in 12.5 μl saline) were delivered for 4 consecutive days, starting on the 9th day after CFA or saline injection. Control animals were injected intrathecally with the same volume of vehicle (n = 8 rats/group). Similarly, CFA rats were injected with 5 μg of MMP-9...

C.-H.Yang, H.-W. Huang, K.-H.Chen, Y.-S.Chen, S.-M.Sheen-Chen and C.-R.Lin. Antinociceptive potentiation and attenuation of tolerance by intrathecal β-arrestin 2 small interfering RNA in rats. Br. J. Anaesth. (2011) doi: 10.1093/bja/aer291.

...Intrathecal β-arrestin 2 (2 μg siRNA per 10 μl per rat) was injected once daily for 3 days. ...10 μl transfection reagent (i-Fect, Neuromics)... 

Alexandre Favereaux, Olivier Thoumine, Rabia Bouali-Benazzouz, Virginie Roques, Marie-Amélie Papon, Sherine Abdel Salam, Guillaume Drutel, Claire Léger, André Calas, Frédéric Nagy and Marc Landry. Bidirectional integrative regulation of Cav1.2 calcium channel by microRNA miR-103: role in pain. The EMBO Journal , (29 July 2011) | doi:10.1038/emboj.2011.249.

...Transfection with seed-mut-miR-103 or scrambled miRNA inhibitor had no effect ..... 10 μl of i-Fect reagent (Neuromics) following Neuromics instructions ...

S. Tumati, W.R. Roeskea, T.M. Largent-Milnesa, T.W. Vanderaha, and EV Varga. Intrathecal PKA-selective siRNA treatment blocks sustained morphine-mediated pain sensitization and antinociceptive tolerance in rats. doi:10.1016/j.jneumeth.2011.04.036.

...transfection reagent (i-Fect; Neuromics, Edina, MN)... 

Emmanuel Deval, Jacques Noël, Xavier Gasull1, Anne Delaunay, Abdelkrim Alloui, Valérie Friend, Alain Eschalier, Michel Lazdunski, and Eric Lingueglia. Acid-Sensing Ion Channels in Postoperative Pain. The Journal of Neuroscience, 20 April 2011, 31(16): 6059-6066; doi: 10.1523/​JNEUROSCI.5266-10.2011.

...were performed 4 and 24 h after surgery. Intrathecal injection of siRNA. Ten microliters of a siRNA (2 mug)/i-Fect (Neuromics) mix was injected intrathecally between the L4 and L5 vertebrae of rats using a Hamilton syringe and a 25 gauge needle...

Shuang-Quan Yu, Donna H. Wang. Intrathecal injection of TRPV1 shRNA leads to increases in blood pressure in rats. DOI: 10.1111/j.1748-1716.2011.02285.x. Copyright © 2011 Scandinavian Physiological Society.

...(Neuromics, NI35150) was used as a transfection reagent. The shRNA/i-Fect complexes in 1:5 ratio (w/v) were used for intrathecal injection. ...

 Wu Fx, Bian Jj, Miao Xr, Huang Sd, Xu Xw, Gong Dj, Sun Ym, Lu Zj, Yu Wf. Intrathecal siRNA against Toll-like receptor 4 reduces nociception in a rat model of neuropathic pain. Int J Med Sci 2010; 7:251-259.

...Rats were randomly divided into four groups with 10 rats in each group: a sham group (Sham surgery + Normal saline, NS), a CCI group (CCI + NS), a MM group (CCI + MM siRNA), and a siRNA group (CCI + TLR4-siRNA). 10 μg SiRNA dissolved in 30 μl i-Fect transfection reagent (Neuromics, Edina, MN, USA) was administered intrathecally once daily for 7 days, starting from 1 day before CCI surgery...

Joseph George, Naren L. Banik and Swapan K. Ray. Survivin knockdown and concurrent 4-HPR treatment controlled human glioblastoma in vitro and in vivo. Neuro-Oncology, doi:10.1093/neuonc/noq079.

...survivin siRNA cDNA was suspended in RNAse free sterile water (25 μg DNA/10 μl) and mixed (1:4 v/v) with i-Fect transfection reagent (Neuromics)...

Philippe Sarret , Louis Doré-Savard1, Nicolas Beaudet. Direct Application of siRNA for In Vivo Pain Research. From: RNA Interference: From Biology to Clinical Applications. Series: Methods in Molecular Biology. Volume: 623; Pub. Date: May-01-2010; Page Range: 383-395; DOI: 10.1007/978-1-60761-588-0_25.

...iFect transfection reagent solution (Neuromics, Minneapolis, MN). .... The selected transfection agent is the cationic lipid i-Fect. ...

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

 ...the siRNAs (2 µg) were solubilized in 10 µl of i-Fect reagent (Neuromics) following Neuromics instructions and published protocol (Luo et al., 2005)...

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...

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...

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. 

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...

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...

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); doi:10.1038/mt.2008.98 

...In this study, we demonstrate the efficacy of 27-mer DsiRNA in reducing the expression of a specific G-protein coupled-receptor (GPCR) gene in rat spinal cord and DRG. Low doses of DsiRNA formulated in i-Fect, when administered by IT injection, induced a sustained reduction in the neurotensin receptor-2 (NTS2) GPCR mRNA and protein levels for 3–4 days. The reduction in NTS2 resulted in the expected behavioral changes in nociception. No apparent toxicity or nonspecific side effects were exhibited during the study period, and our results overall highlight the feasibility of using DsiRNA in pain research... 

X.-W. Dong, S. Goregoaker, H. Engler, X. Zhou, L. Mark, J. Crona, R. Terry, J. Hunter and T. Priestley. Small interfering RNA-mediated selective knockdown of NaV1.8 tetrodotoxin-resistant sodium channel reverses mechanical allodynia in neuropathic rats. Neuroscience Volume 146, Issue 2, 11 May 2007, Pages 812-821  

...For in vivo studies, siRNA stock solution (1 mg/ml) was prepared using siRNA suspension buffer (Qiagen). The solution was then heated to 90 °C for 1 min followed by 60 min incubation at 37 °C. Aliquots were stored at -20 °C. On the day of the injection, an aliquot of the siRNA was thawed on ice and then mixed with the transfection reagent, iFect (Neuromics Antibodies, Northfield, MN, USA), to yield a final concentration of 0.2 ug/ ul. A total of 100 ul of the siRNA solution or vehicle (20 ul of suspension buffer in 80 ul of iFect; 1:4 v/v) was locally delivered to lumbar DRGs through implanted epidural catheter by slow injection. The catheter was flushed with 15 ul sterile saline...

Luo MC, Zhang DQ, Ma SW, Huang YY, Shuster SJ, Porreca F, Lai J (2005). An efficient intrathecal delivery of small interfering RNA to the spinal cord and peripheral neurons. Molecular Pain 2005, 1:29.

Priti Kumar, Sang Kyung Lee, Premlata Shankar, N. Manjunath. A Single siRNA Cynthia C. Tsui, Stuart J. Shankland and Brian A. Pierchala. Glial Cell Line–Derived Neurotrophic Factor and Its Receptor Ret Is a Novel Ligand-Receptor Complex Critical for Survival Response during Podocyte Injury. doi: 10.1681/ASN.2005080835

...Ret small interference RNA (siRNA) knockdown was performed by using transient transfection of pooled functionally validated Ret siRNA (SMARTpool mouse RET siRNA; Dharmacon, Lafayette, CO). HSMP cells that were differentiated for 10 to 12 d were maintained at 10% FBS/RPMI as described above and transfected using the i-Fect siRNA transfection reagent (Neuromics, Edina, MN). For determination of the transfection efficiency, a Texas Red–labeled siRNA (siGLO RISCFree siRNA; Dharmacon) was co-transfected with Ret siRNA and visualized using fluorescence microscopy. For control siRNA samples, identical conditions were used with the substitution of siGLO-RISCFree siRNA for Ret siRNA. For determination of the efficiency of Ret knockdown, Western analysis for Ret was performed on WCL from cells 24 to 96 h after the transfection. Several concentrations of Ret siRNA (40, 60, and 100 nM) were tested to determine optimal knockdown conditions. For apoptosis assays, podocytes were exposed to UV-C or PA (40 g/ml) on days 3 to 4 after transfection of Ret siRNA or control siRNA (100 nM). Apoptosis was measured by counting podocytes with Hoechst-positive pyknotic nuclei 3 h after UV and 5 h after exposure to PA...

n-FectTMTransfection Kits

Amy N. Sussman, Tong Sun, Ronald M. Krofft, and Raghu V. Durvasula. SPARC Accelerates Disease Progression in Experimental Crescentic Glomerulonephritis. Am. J. Pathol., May 2009; 174: 1827 - 1836.

...empty plasmid inserted into the NHe1/BamH1 sites of pcDNA3.1/Zeo () vector (Invitrogen, Carlsbad, CA) using N-Fect reagent (Neuromics, Edina, MN) according to manufacturers instructions. Transfected cells were selected in RPMI 1640 media supplemented with...

Hidetaka Sadanari, Junji Tanaka, Zhuan Li, Rie Yamada, Keiko Matsubara and Tsugiya Murayama.Proteasome inhibitor differentially regulates expression of the major immediate early genes of human cytomegalovirus in human central nervous system-derived cell lines. doi:10.1016/j.virusres.2009.01.010 .

King-Ho Cheung, Diana Shineman, Marioly Müller, César Cárdenas, Lijuan Mei1, Jun Yang, Taisuke Tomita, Takeshi Iwatsubo, Virginia M.-Y. Lee, and J. Kevin Foskett. Mechanism of Ca2+ Disruption in Alzheimer's Disease by Presenilin Regulation of InsP3 Receptor Channel Gating. doi:10.1016/j.neuron.2008.04.015

...pIRES2-EGFP empty vector by n-Fect reagent (Neuromics)...

Griffin SV, Hiromura K, Pippin J, Petermann AT, Blonski MJ, Krofft R, Takahashi S, Kulkarni AB, Shankland SJ (2004). Cyclin-Dependent Kinase 5 Is a Regulator of Podocyte Differentiation, Proliferation, and Morphology. Am J Pathol. 165:1175-1185.

p-Fect™Transfection Kits

Hunnicutt BJ , Chaverra M , George L , Lefcort F , 2012 IKAP/Elp1 Is Required In Vivo for Neurogenesis and Neuronal Survival, but Not for Neural Crest Migration. PLoS ONE 7(2): e32050. doi:10.1371/journal.pone.0032050

...Dorsal root ganglia were dissected from Embryonic day 5–9.5 chick embryos (E5–9.5) and dissociated by incubation in 0.25% trypsin-EDTA (Gibco) for 7 min at 37 C followed by trituration through fire–pulled glass pipettes. The culture media consisted of Neurobasal medium (Invitrogen) supplemented with B27 (1X,Invitrogen), Glutamax (1X,Invitrogen), Hybrimax Antibiotic\Antimicotic (1:100, Sigma), NGF (10 ng/ml, gift from Dr. Thomas Large). Cells were plated on 8-well Nunc glass chamber slides that were coated with poly-D-lysine (1:100, Sigma) and laminin 20 ug/ml (Gibco). Approximately equal numbers of cells (52,500) were plated per well. Immediately after plating, cells were transfected with IKBKAP-7.4 shRNA or control shRNA via pn-Fect (Neuromics, PN3375). Several ratios of pnfect:DNA were tested with the optimum obtained 1.84:1. The cells were then cultured for approximately 29 h at 37 C, 5.5% C02. After incubation culture cells were fixed and inmunostained as previously described [24]. To determine whether IKBKAP shRNAs altered cell proliferation and/or neuronal differentiation in dissociated DRG cultures, Brdu was added to the cultures and the cells were fixed 24 hrs later (as described in [24]). The number of GFP+/BrdU+ or GFP+/Tuj-1+ cells were quantified for each experiment, and a ratio comparing control vs. IKBKAP shRNAs for each experiment determined. For the BrdU+ experiment, a total of 556 GFP+/control shRNA transfected cells were counted and 357 GFP+/IKBKAP shRNA transfected cells in 3 separate experiment. For determining neuronal differentiation, a total of 2866 GFP+/control shRNA transfected cells and 2913 IKBKAP shRNA transfected cells were counted, over 3 separate experiments...

Shih-Kuo Chen, Gladys Y.-P. Ko, and Stuart E. Dryer. Somatostatin Peptides Produce Multiple Effects on Gating Properties of Native Cone Photoreceptor cGMP-Gated Channels That Depend on Circadian Phase and Previous Illumination. J. Neurosci., Nov 2007; 27: 12168 - 12175 ; doi:10.1523/JNEUROSCI.3541-07.2007.

...Transfection was performed using pn-Fect from Neuromics (Edina, MN). Briefly, retinal cells were grown for 4 d in LD cycles, until 24 h before analysis. At that time, coverslips with attached retinal cells were transferred to 400 µl of serum-reduced culture medium (Optimem; Invitrogen, Carlsbad, CA) in 24-well plates supplemented with 100 µl of solution containing pn-Fect with PLC{delta}-PH-GFP expression vector incorporated according to the manufacturer's instructions. In a series of pilot experiments, we determined that the maximum transfection efficiency occurred with a pn-Fect/DNA ratio of 1.4/1... 

i-Fect in vivo!
USPTO Patent Application 20080031911-Smad Signaling and Neuro-regeneration
United States Patent 20070105806
Pub from The Journal of Neuroscience
June 13, 2007, 27(24):6363-6373

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i-Fect in vivo!
USPTO Patent Application 20080031911-Smad Signaling and Neuro-regeneration United States Patent 20070105806 Pub from The Journal of Neuroscience June 13, 2007, 27(24):6363-6373