Thursday, May 24, 2012

i-Fect, Troy, Microglia and Glioblastomas

Neuromics' i-FectTM delivers siRNA to Microglia in Culture.

I have reported success with using i-Fect both in vitro and in vivo. I am excited about the results featured in this publication as Neurons and Glia are particulary tough to transfect with siRNA. I congratulate the researchers for successfully knocking down the Troy gene in vitro: 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.

Highlights: "We demonstrate that inhibition of TROY expression in microglia by siRNA transfection significantly inhibits microglial migration towards CNS-1 cells similar to 10 µM propentofylline treatment. These results identify TROY as a novel molecule expressed in microglia, involved in their migration and targeted by propentofylline. Furthermore, these results describe a signaling molecule that is differentially expressed between microglia and macrophages in the tumor microenvironment."

Protocol: 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 [J Am Soc Nephrol 17: 1543–1552]. 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.

Images: (A) Western blot demonstrating decreased TROY expression in microglia cultured with CNS-1 conditioned media and treated with TROY siRNA. (B) Microglia were treated with TROY siRNA, and then migrated towards CNS-1 cells. Migration of microglia in response to CNS-1 cells is significantly decreased compared to media (* = p<0.05).

Check out all pubs referencing use of i-Fect.