Delivery of siRNA, miRNA, shRNA and Plasmids Guaranteed

Potent and Frequently Published Transfection Solutions


We have proven and frequently published transfection solutions. 
Powerful punch and versatility are now needed more than ever with the 
hyper growth in gene manipulation technologies like Sleeping Beauty^TM
and CRISPR-Cas9.

We have published examples of the use of our solutions for delivering siRNA, miRNA, 
shRNA and plasmids both in vitro and in vivo. here are some of your options.
i-Fect ™ -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.
p-Fect™ -Designed to delivery plasmids, DNA or 
RNA to hard to transfect Cell Lines.

pn-Fect™ -The latest advance in transfection 
for primary neuronal cells. 
This unique reagent provides ultra-high plasmid DNA delivery 
efficiencies and low cytotoxicity compared to competitive reagents.
Here's a recent i-Fect Publication:
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
 

Figure: Knockdown of Id2 attenuated mechanical allodynia and thermal 

hyperalgesia in CCI rats. (a and b) PWT and PWL were measured 1 day before 

CCI and 1, 3, 7, and 14 days after intrathecal administration of shRNA-Id2.

If you are looking for transfection solutions, do not hesitate to contact me @ direct phone: 612-801-1007 or pshuster@neuromics.com. Thank you, Pete Shuster, CEO and Owner, Neuromics

Neuromics' Transfection Kits-Genes Studied

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

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

We can now add GPNMB to this list: Lili Hou, Yanfeng Zhang, Yong Yang, Kai Xiang, Qindong Tan, Qulian Guo. Intrathecal siRNA Against GPNMB Attenuates Nociception in a Rat Model of Neuropathic Pain. Journal of Molecular Neuroscience. July 2014...Ten micrograms of siRNA1- GPNMB dissolved in 30 μl i-Fect transfection reagent (Neuromics, Edina, MN, USA) was administered intrathecally once daily for 7 days, starting from 1 day before CCI surgery...

Abstract: Neuropathic pain is characterized by hyperalgesia, allodynia, and spontaneous pain. Recent studies have shown that glycoprotein nonmetastatic melanoma B (GPNMB) plays a pivotal role in neuronal survival and neuroprotection. However, the role of GPNMB in neuropathic pain remains unknown. The aim of the present study was to assess the role of GPNMB in neuropathic pain. In cultured spinal cord neurons, we used two small interfering RNAs (siRNAs) targeting the complementary DNA (cDNA) sequence of rat GPNMB that had potent inhibitory effects on GPNMB, and siRNA1-GPNMB was selected for further in vivo study as it had the higher inhibitory effect. After sciatic nerve injury in rats, the endogenous level of GPNMB was increased in a time-dependent manner in the spinal cord. Furthermore, the intrathecal injection of siRNA1-GPNMB inhibited the expression of GPNMB and pro-inflammatory factors (TNF-α, IL-1β, and IL-6) and alleviated mechanical allodynia and thermal hyperalgesia in the chronic constriction injury (CCI) model of rats. Taken together, our findings suggest that siRNA against GPNMB can alleviate the chronic neuropathic pain caused by CCI, and this effect may be mediated by attenuated expression of TNF-α, IL-1β, and IL-6 in the spinal cord of CCI rats. Therefore, inhibition of GPNMB may provide a novel strategy for the treatment of neuropathic pain.

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

pn-Fect and Transfection of DRG cultures

In Vitro Gene expression analysis assays are essential for understanding how up or down regulation of  related target proteins could result in pathologies. Dorsal Root Ganglion (DRG), Neuronal and Glial Cultures have proven hard to transfect as as many transfection reagents are toxic to these cells. It is important for the study of neuro-diseases that researchers have tools and methods that enable success.

In this study, researchers successfully transfect DRG cultures with IKAP-shRNA using our pn-Fect kit. The own regulation of IKAP in these cultures support findings that helped explain the potential pathology of Familial Dysautonomia (FD; Hereditary Sensory Autonomic Neuropathy; HSAN III): 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.

Cell culture: 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.



Images: IKAP regulates neuronal differentiation in the DRG. Reduction in IKAP leads to increased numbers of neurons in the immature DRG (A–C). Embryos at St. 12 were transfected with either control shRNAs or IKBKAP shRNAs and analyzed at St 24/25. Embryos were sectioned, and immunolabled with the neuronal markers Tuj-1 or Ben and the percentage of GFP+ neurons determined. Significantly more IKBKAP shRNA transfected DRG precursor cells differentiated into neurons (arrows in B; IKBKAP shRNA 7.4; n = 3 embryos; p = 0.002; IKBKAP shRNA 1.6 & 4.5, n = 3 embryos; p = 0.001) than Control shRNA transfected DRG precursor cells (n = 5 embryos). (D–H) IKBKAP shRNA-transfected DRG precursor cells (n = 3 embryos; 252 cleaved-Caspase 3+ cells counted) were also more likely to die by apoptosis (compare D & E to F & G) than control shRNA-electroporated cells (n = 3 embryos; 117 cleaved caspase-3+ cells counted). (H) The number of cleaved Caspase 3+ cells was quantified in DRG on both the transfected side of the embryo and the non-transfected side of the embryo and a ratio determined. Significantly more cleaved-Caspase 3+ cells were present in the transfected DRG of IKBKAP shRNA transfected embryos than in the transfected DRG in embryos transfected with control shRNAs; p = 0.006. (I–N). Over-expression of the c-terminus of IKAP prevents neural crest cells from coalescing with the DRG (I, K, L; p = 0.004) but the few that do join, tend not to differentiate into neurons (p = 0.007; J, M, N). Embryos were transfected with either a construct driving expression of the c-terminus of chicken IKAP with a His tag (CT-IKAP-His; L,N) or a control, His-tagged construct (K, M) and analyzed at St. 21. The location and neuronal identity of transfected cells was determined in 3 embryos for each treatment; Control His-plasmid: n = 343 transfected cells counted; CT-IKAP-His: n = 278 transfected cells counted. Statistical analysis by Student t-test. doi:10.1371/journal.pone.0032050.g006. 


Check out our transfection reagents and capabilities. We will continue to post publications and data that support and add to our capabilities.