Friday, August 19, 2016

Delivering miRNA in vivo

More Transfection Success! Great Research Tools!

Our i-FectTM  Transfection Kit is used to study Epigenetics and pain. Here's yet another example: 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...
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.

Finding like these could pave the way for an miRNA like therapy for pain.

Monday, June 20, 2016

i-Fect used to Study Epigenetics in Nerve Injury

i-Fect used In Vivo for Study

Researchers use our i-FectTM to effectively deliver miR-126 in vivo to modulate Methyl-CpG-binding protein 2 (MeCP2).

MeCP2 regulates gene expression through activation, repression and chromatin remodeling. Mutations in MeCP2 cause Rett syndrome, and these patients display impaired nociception. 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. 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 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...

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).

Conclusions: 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.

Thursday, March 24, 2016

Epigenetics and Pain Research

i-Fect Used to Study Impacts

Our i-Fect siRNA, miRNA and shRNA Trasfection Kit was recently used to study the impact of G9a-specific siRNA (AGUAACGGGCAUCAAUGC) on Mu Opioid Receptors: 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...

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).

Summary: 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.

Wednesday, January 20, 2016

Delivery of siRNA, miRNA, shRNA and Plasmids Guaranteed

Potent and Frequently Published Transfection Solutions

Gene Tools
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 BeautyTM
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 Thank you, Pete Shuster, CEO and Owner, Neuromics

Monday, November 16, 2015

Kv Channels and Acute to Chronic Pain Transistion

i-Fect TMis a Proven Tool for Gene Manipulation in Studying All Types of Pain

I previously posted on use of our i-Fect Transfection Kit to silence Kv Channels Receptors. This has enabled researchers to study the role of these receptors in vitro and in vivo (see:i-Fect™ Delivers Your siRNA Payload).

Sample Data

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

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 publications referencing use of our Transfection Kits to over 45: 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.

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."

I will continue to post here new and unique solutions and related referencing for our Gene Expression Analysis Tools.

Monday, August 3, 2015

Introducing Sleeping Beauty

New Technology for Gene Transfer From Delivery to Stable Expression
Neuromics has a successful track record of helping our clients delivery siRNA, miRNA, Plasmids and other oligos in vitro and in vivo with our Transfection Kits...But my vision with our cell based assay solutions 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:
Gene Transfer and Expression Products Leveraging the Sleeping Beauty Technology

Images: B-MoGen Transposon exhibiting stable expression of five fluorescent genes
Advantages of Sleeping Beauty Transposon System:
· Delivery method is time and cost effective compared to lentiviral delivery.
· Increased cargo-capacity when compared to lentiviral delivery.
· Safest insertion profile of all gene transfer methods.
· Commonly integrated as a single copy.
Custom vector design and assembly, including multi-gene (up to 6) vectors.
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 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.

Tuesday, April 7, 2015

Silencing Cytokines in-vivo with i-Fect

Knocking Down Cytokines to Study Pain Response

We have many unique applications published by researchers using our Transfection Kits in vitro and in vivo. Here researchers simultaneously silence 3 immune/inflammatory response cytokines 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.

Protocol: 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.

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.
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.

We will continue to post new applications and methods published by researchers using our Transfection Kits.