5 g/ml totally free siRNA resolution was set as 100 . The level of
five g/ml cost-free siRNA option was set as one hundred . The amount of siRNA offered to interact with all the SYBR Green I is expressed as a percentage of your control.two.7. Luciferase activity MCF-7-Luc cells had been ready by plating cells in a 6-well plate 24 h before every single experiment. For transfection, every single lipoplex of 200 pmol Luc siRNA, Luc-siRNA-Chol, Cont siRNA or Cont siRNA-Chol was diluted in two ml of medium supplemented with 10 FBS and after that the mixture was added into the cells. Lipofectamine RNAiMax lipoplex (Invitrogen Corp.) was prepared in accordance with the manufacturer’s protocol. Forty-eight hours following the transfection, luciferase activity was measured as counts per s (cps)/ g protein using the luciferase assay system (Pica gene luciferase assay kit; Toyo Ink Mfg. Co., Ltd.) and BCA reagent (Pierce, Rockford, IL, USA), as previously reported [11].Y. Hattori et al. / Benefits in Pharma Sciences 4 (2014) 12.8. Agglutination assay Agglutination assay was performed based on the approach described inside a prior report [5]. Briefly, erythrocytes have been collected from mouse blood at 4 C by centrifugation at 300g for three min and resuspended in PBS as a two (v/v) stock suspension of erythrocytes. The anionic polymer-coated NOD1 Storage & Stability lipoplexes of 2 g of Cont siRNA or siRNAChol were added to one hundred L of erythrocyte suspension and then incubated for 15 min at 37 C. The sample was placed on a glass plate and agglutination was observed by microscopy. 2.9. Biodistribution of anionic polymer-coated lipoplexes in mice All animal experiments had been performed with approval in the Institutional Animal Care and Use Committee of Hoshi University. Cationic and anionic polymer-coated lipoplexes of 50 g of Cy5.5siRNA or Cy5.5-siRNA-Chol were intravenously administered by way of lateral tail veins into female BALB/c mice (7 weeks of age; Sankyo Lab. Service Corp., Tokyo, Japan). One hour just after injection, the mice had been sacrificed, plus the tissues have been frozen on dry ice and sliced at 16 m. The localization of Cy5.5-siRNA was examined making use of an Eclipse TS100F microscope (Nikon, Tokyo, Japan). 2.10. Knockdown of liver-specific ApoB mRNA in vivo Anionic polymer-coated lipoplexes of 50 g of Cont siRNA-Chol or ApoB siRNA-Chol were intravenously administered via lateral tail veins into mice. At 24 h post-injection, mice have been fasted for 24 h. At 48 h post-injection, mice were sacrificed by cervical dislocation as well as the liver was removed for analysis. Total RNA was isolated in the liver employing the PKCθ site NucleoSpin RNA II (MachereyNagel, Germany). Mouse ApoB cDNA was amplified using the primers ApoB-FW, 5 -TTCCAGCCATGGGCAACTTTACCT-3 , and ApoBRW, five -TACTGCAGGGCGTCAGTGACAAAT-3 , as previously reported [12]. Mouse -actin cDNA was amplified working with the primers actin-FW, five -TGTGATGGTGGGAATGGGTCAG-3 , and -actin-RW, five TTTGATGTCACGCACGATTTCC-3 , as previously reported [13]. Quantitative RT-PCR was performed using the iCycler MyiQ detection program (Bio-Rad Laboratories, Hercules, CA, USA) plus the SYBR Green I assay TM (iQ SYBER Green Supermix, Bio-Rad Laboratories), as previously described [13]. Samples have been run in triplicate plus the mRNA expression levels of ApoB had been normalized to the volume of -actin mRNA inside the identical sample. A distinction of 1 cycle was thought of to represent a 2-fold change in gene expression. two.11. Serum cholesterol level PGA-coated lipoplexes of 50 g of ApoB siRNA-Chol had been intravenously administered through lateral tail veins into mice. At 24 h postinjection, mice have been fasted for 24 h.
