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PureFection Transfection Reagent

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[#LV750A-1] PureFection Transfection Reagent

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(1) Effects of HMGA2 gene downregulation by siRNA on lung carcinoma cell migration in A549 cell lines.[TOP]

Pubmed ID :30317663
Publication Date : //
Although there are multiple treatments for lung cancer, the death rate of this cancer remains high because of metastasis in earlier stages. So a novel treatment for overcoming metastasis is urgently needed. Overexpression of high-mobility group AT-hook 2 (HMGA2), a nonhistone chromosomal protein has been observed in metastatic cancers. So, we suggested that HMGA2 upregulation may play a critical role in treating lung cancer.

Authors : Naghizadeh Sanaz, Mansoori Behzad, Mohammadi Ali, Kafil Hossein Samadi, Mousavi Zohreh, Sakhinia Ebrahim, Baradaran Behzad,



(2) Mechanism of piR-DQ590027/MIR17HG regulating the permeability of glioma conditioned normal BBB.[TOP]

Pubmed ID :30305135
Publication Date : //
The blood-brain barrier (BBB) strongly restricts the entry of anti-glioma drugs into tumor tissues and thus decreases chemotherapy efficacy. Malignant gliomas are highly invasive tumours that use the perivascular space for invasion and co-opt existing vessels as satellite tumor form. Because regulation of the effect of noncoding RNA on BBB function is attracting growing attention, we investigated the effects of noncoding RNA on the permeability of glioma conditioned normal BBB and the mechanism involved using PIWI-associated RNA piR-DQ590027 as a starting point.

Authors : Leng Xue, Ma Jun, Liu Yunhui, Shen Shuyuan, Yu Hai, Zheng Jian, Liu Xiaobai, Liu Libo, Chen Jiajia, Zhao Lini, Ruan Xuelei, Xue Yixue,



(3) Systematic Screening of Commonly Used Commercial Transfection Reagents towards Efficient Transfection of Single-Stranded Oligonucleotides.[TOP]

Pubmed ID :30297632
Publication Date : //
Non-viral vector-mediated transfection is a core technique for in vitro screening of oligonucleotides. Despite the growing interests in the development of oliogonucleotide-based drug molecules in recent years, a comprehensive comparison of the transfection efficacy of commonly used commercial transfection reagents has not been reported. In this study, five commonly used transfection reagents, including Lipofectamine 3000, Lipofectamine 2000, Fugene, RNAiMAX and Lipofectin, were comprehensively analyzed in ten cell lines using a fluorescence imaging-based transfection assay. Although the transfection efficacy and toxicity of transfection reagents varied depending on cell types, the toxicity of transfection reagents generally displayed a positive correlation with their transfection efficacy. According to our results, Lipofectamine 3000, Fugene and RNAiMAX showed high transfection efficacy, however, RNAiMAX may be a better option for majority of cells when lower toxicity is desired. The transfection efficacy of Lipofectamine 2000 was compromised by its high toxicity, which may adversely affect its application in most cells. We firmly believe that our findings may contribute to the future In vitro delivery and screening of single-stranded therapeutic oligonucleotides such as antisense oligonucleotides, antimiRs, and DNAzymes.

Authors : Wang Tao, Larcher Leon M, Ma Lixia, Veedu Rakesh N,



(4) Quantitative colocalization analysis of DNA delivery by PEI-mediated cationic polymers in mammalian cells.[TOP]

Pubmed ID :30295315
Publication Date : //
Although cationic polymers are widely used for DNA delivery, the relationship between the properties of the formed complexes and their biological activity is not fully understood. Here, we propose a novel procedure consisting of superresolved images coupled with quantitative colocalization to analyse DNA release in living cells. This work compares the different workflows available in a quantitative colocalization study of DNA delivery using polyethylenimine as transfection reagent. A nimble workflow with deconvolution in three-dimensional images was developed. Among the different colocalization coefficients, Manders' colocalization coefficient was the best to track the complexes. Results showed that DNA/polyethylenimine complexes were tightly interacting at the time of transfection and their disassembly was observed between 2 and 10 h after their uptake. Heterogenicity was found in the intracellular fate of each complex. At 24 h, some complexes were still present underneath the nuclear envelope. Overall, this study opens the door for particle tracking assessment with three-dimensional imaging at intracellular level.

Authors : González-Domínguez I, Cervera L, Gòdia F, Roldán M,



(5) High-Throughput DNA Plasmid Transfection Using Acoustic Droplet Ejection Technology.[TOP]

Pubmed ID :30290128
Publication Date : //
The Labcyte Echo acoustic liquid handler allows accurate droplet ejection at high speed from a source well plate to a destination plate. It has already been used in various miniaturized biological assays, such as quantitative PCR (q-PCR), quantitative real-time PCR (q-RT-PCR), protein crystallization, drug screening, cell dispensing, and siRNA transfection. However, no plasmid DNA transfection assay has been published so far using this dispensing technology. In this study, we evaluated the ability of the Echo 550 device to perform plasmid DNA transfection in 384-well plates. Due to the high throughput of this device, we simultaneously optimized the three main parameters of a transfection process: dilution of the transfection reagent, DNA amount, and starting DNA concentration. We defined a four-step protocol whose optimal settings allowed us to transfect HeLa cells with up to 90% efficiency and reach a co-expression of nearly 100% within transfected cells in co-transfection experiments. This fast, reliable, and automated protocol opens new ways to easily and rapidly identify optimal transfection settings for a given cell type. Furthermore, it permits easy software-based transfection control and multiplexing of plasmids distributed on wells of a source plate. This new development could lead to new array applications, such as human ORFeome protein expression or CRISPR-Cas9-based gene function validation in nonpooled screening strategies.

Authors : Colin Béatrice, Deprez Benoit, Couturier Cyril,



(6) Synthesis, characterization and evaluation of transfection efficiency of dexamethasone conjugated poly(propyleneimine) nanocarriers for gene delivery#.[TOP]

Pubmed ID :30270694
Publication Date : //
Polypropylenimine (PPI), a cationic dendrimer with defined structure and positive surface charge, is a potent non-viral vector. Dexamethasone (Dexa) conveys to the nucleus through interaction with its intracellular receptor.

Authors : Malaekeh-Nikouei Bizhan, Rezaee Mehdi, Gholami Leila, Sanjar Mousavi Naghmeh, Kazemi Oskuee Reza,



(7) DOTAP, a lipidic transfection reagent, triggers Arabidopsis plant defense responses.[TOP]

Pubmed ID :30255355
Publication Date : //
DOTAP triggers Arabidopsis thaliana immunity and by priming the defense response is able to reduce bacterial pathogen attack. DOTAP is a cationic lipid widely used as a liposomal transfection reagent and it has recently been identified as a strong activator of the innate immune system in animal cells. Plants are sessile organisms and unlike mammals, that have innate and acquired immunity, plants possess only innate immunity. A key feature of plant immunity is the ability to sense potentially dangerous signals, as it is the case for microbe-associated, pathogen-associated or damage-associated molecular patterns and by doing so, trigger an active defense response to cope with the perturbing stimulus. Here, we evaluated the effect of DOTAP in plant basal innate immunity. An initial plant defense response was induced by the cationic lipid DOTAP in the model plant Arabidopsis thaliana, assessed by callose deposition, reactive oxygen species production, and plant cell death. In addition, a proteomic analysis revealed that these responses are mirrored by changes in the plant proteome, such as up-regulation of proteins related to defense responses, including proteins involved in photorespiration, cysteine and oxylipin synthesis, and oxidative stress response; and down-regulation of enzymes related to photosynthesis. Furthermore, DOTAP was able to prime the defense response for later pathogenic challenges as in the case of the virulent bacterial pathogen Pseudomonas syringae pv. tomato. Disease outcome was diminished in DOTAP-pre-treated leaves and bacterial growth was reduced 100 times compared to mock leaves. Therefore, DOTAP may be considered a good candidate as an elicitor for the study of plant immunity.

Authors : Grandellis Carolina, Garavaglia Betiana S, Gottig Natalia, Lonez Caroline, Ruysschaert Jean-Marie, Ottado Jorgelina,



(8) High Throughput Transfection of HEK293 Cells for Transient Protein Production.[TOP]

Pubmed ID :30242687
Publication Date : //
Transient transfection of mammalian cells is used in the biotechnology industry to quickly supply recombinant protein for research and large molecule drug development. Here, we describe a method for high throughput transient transfection of Human Embryonic Kidney 293 (HEK293) cells in 30 mL tubespins using polyethylenimine (PEI) as a transfection reagent. An automated liquid handler can be used to perform pipetting steps for transfecting batches of 96 tubespins, and septa in the tubespin caps allow for rapid processing without decapping. The addition of valproic acid (VPA) to transfection cultures enhances recombinant protein production. The thawing and passaging operations for HEK293 cultures to source the transient transfections are also described.

Authors : Arena Tia A, Harms Peter D, Wong Athena W,



(9) Transient Gene Expression in Suspension HEK293-EBNA1 Cells.[TOP]

Pubmed ID :30242676
Publication Date : //
Transient gene expression in human embryo kidney 293 (HEK293) cells is an established approach for the rapid production of large amounts of recombinant proteins (r-proteins). Milligram to gram quantities of r-proteins can be typically obtained within less than 10 days following transfection. In this chapter, we describe a simple and robust transfection process of suspension-growing human embryo kidney 293 cells using two commercially available serum-free media and polyethylenimine as the transfection reagent. This chapter provides examples for the production and purification of a his-tagged recombinant protein and two monoclonal antibodies.

Authors : L'Abbé Denis, Bisson Louis, Gervais Christian, Grazzini Eric, Durocher Yves,



(10) Optimization of the Linker Length of Mannose-Cholesterol Conjugates for Enhanced mRNA Delivery to Dendritic Cells by Liposomes.[TOP]

Pubmed ID :30233368
Publication Date : //
Liposomes (LPs) as commonly used mRNA delivery systems remain to be rationally designed and optimized to ameliorate the antigen expression of mRNA vaccine in dendritic cells (DCs). In this study, we synthesized mannose-cholesterol conjugates (MP-CHs) by click reaction using different PEG units (PEG, PEG, and PEG) as linker molecules. MP-CHs were fully characterized and subsequently used to prepare DC-targeting liposomes (MP-LPs) by a thin-film dispersion method. MP-LPs loaded with mRNA (MP-LPX) were finally prepared by a simple self-assembly method. MP-LPX displayed bigger diameter (about 135 nm) and lower zeta potential (about 40 mV) compared to MP-LPs. The transfection experiment on DC2.4 cells demonstrated that the PEG length of mannose derivatives had significant effect on the expression of GFP-encoding mRNA. MP-LPX containing MP-CH can achieve the highest transfection efficiency (52.09 ± 4.85%), which was significantly superior to the commercial transfection reagent Lipo 3K (11.47 ± 2.31%). The optimal DC-targeting MP-LPX showed an average size of 132.93 ± 4.93 nm and zeta potential of 37.93 ± 2.95 mV with nearly spherical shape. Moreover, MP-LPX can protect mRNA against degradation in serum with high efficacy. The uptake study indicated that MP-LPX enhanced mRNA expression mainly through the over-expressing mannose receptor (CD206) on the surface of DCs. In conclusion, mannose modified LPs might be a potential DC-targeting delivery system for mRNA vaccine after rational design and deserve further study on the delivery profile and anti-tumor efficacy.

Authors : Wang Fazhan, Xiao Wen, Elbahnasawy Mostafa A, Bao Xingting, Zheng Qian, Gong Linhui, Zhou Yang, Yang Shuping, Fang Aiping, Farag Mohamed M S, Wu Jinhui, Song Xiangrong,