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1、1About three fluorescence marker of the short double-stranded RNA in vitro transfection characteristics ComparativeAuthor: Ma Yun, COMMITTED, Ruled, Wen An Min, Gao Jun, ZHOU Can-quan, Chen Gu Abstract Objective: To compare three different fluorescently labeled short double-stranded RNA (siRNA) tran
2、sfection in vitro characteristics: using liposomes LipofectamieTM2000 transfected three fluorescence marker siRNA into mouse embryonic fibroblast cell line NIH 3T3 flow cytometer observed transfection efficiency the ordinary inverted fluorescence microscope quenching, laser scanning confocal microsc
3、ope to observe the distribution within the cell. results: three fluorescent under the same conditions of transfection siRNA speed into the cell, intracellular different positioning, different transfection efficiency, three fluorescently labeled antifade have different capacities. Conclusion: fluores
4、cence-labeled siRNA can play a good tracer reference as the distribution of the fluorescence of siRNA and transfection efficiency with the fluorescent-labeled 2siRNA to take into account the selection of fluorescently labeled siRNA with no fluorescently labeled siRNA differences. Keywords fluorescen
5、tly labeled siRNA transfection Abstract AIM: To compare the transfection character of three different fluorescently labeled small interfering RNA (siRNA) in vitro. METHODS: Three fluorescently labeled siRNA were transfected with LipofectamieTM2000 into NIH 3T3 cells for moni toring their transfectio
6、n efficiency with flow cytometry, tracking their delivery with confocal microscopy and checking their photostability with fluorescent microscope. RESULTS: These three fluorescently labeled siRNA varied in intracellular distribution, transfection efficiency and quench speed. CONCLUSION: The fluoresce
7、nce labeled siRNA helps to tracking the delivery of siRNA in cells. The difference between fluorescently labeled siRNA and unlabeled siRNA should be considered when the fluorescently labeled siRNA is used to study the distribution and transfection efficiency of siRNA. 3Keywords fluorescence label, s
8、mall interfering RNA, transfection 0 Introduction SiRNA can be chemically synthesized efficiently and specifically inhibit the expression of target genes, not only is a powerful tool for analysis of gene function, and is expected to become new drugs 1, but, the delivery efficiency is low and in vivo
9、 efficacy problem is still the bottleneck of its application, therefore, the depth to study the distribution and dynamics of the process in the body gradually attention 2. using fluorescence microscopy, flow cytometry, laser scanning confocal microscopy and other methods to detect fluorescently labe
10、led siRNA 3, to observe the uptake, distribution and metabolism in order to infer no fluorescently labeled siRNA transfection efficiency and distribution. however although there are a variety of commercialization of fluorescently labeled siRNA silencing effect alternative, but their distribution and
11、 transfection efficiency is consistent yet rarely reported. This study compared three commonly used fluorescent labeled siRNA transfection liposomal transfection efficiency, fluorescence quenching and intracellular 4distribution characteristics, in order to explore the issues that need to pay attent
12、ion to the application of fluorescent labeled siRNA. 1 Materials and methods 1.1 Materials The three fluorescent siRNA: BLOCK iTTM fluorescent oligomer (sequence undisclosed) were purchased from Invitrogen Corporation, FAM the no target siRNA (sense strand 5 UUCUCCGAACGUGUCACGU 3, FAM labeled at the
13、 5 end) were purchased from Guangzhou Rui Bo , Alexa Fluor 546 negative control siRNA (target sequence 5 AATTCTCCGAACGTGTCACGT 3 sense strand 3 end of the 3 marked the Alexa Fluor 546) was purchased from Qiagen first two ready-to-use, the latter join annealing buffer in accordance with the manual me
14、thod , 90 denaturation and annealing, repackaging stored at -20 C for three fluorescent siRNA stock solution concentration was 20 mol / L. the cryopreservation of mouse embryonic fibroblast cell line NIH3T3 cell library by the Experimental Animal Center of Sun Yat-sen University high glucose DMEM me
15、dium and DPBS were purchased from Gibco BRL; fetal bovine serum 5was purchased from Hangzhou Evergreen Institute of Engineering Materials liposomes LipofectamineTM the 2000 Opti MEM low serum medium were purchased from Invitrogen of, Hochest33342 purchased from Sigma (USA) other biochemical reagents
16、 are imported or domestic analytical reagents. 1.2 Methods 1.2.1 Cell culture and transfection NIH3T3 cells were routinely cultured in 100 mL / L fetal calf serum high glucose DMEM medium without antibiotics per well of 4 104 cells were seeded 24-well plates, 24 h after cell confluence of 40%. Use LipofectamineTM 2000 transfection fluorescence siRNA, in accordance with the manual operation. siRNA when the transfection efficiency of d
