Hi all,

I have nanoparticles that encapsulate and deliver to cells a ssRNA of 21 nucleotides (that is supposed to act as a microRNA). I am going to transfect those nanoparticles into NCTC 929 fibroblasts. For transfection assays that use plasmid DNA, the incubation time is normally 48 hours after the addition of the plasmid DNA to the cell culture. But in my case, since I am using single-stranded RNA, it will not need to enter in the nucleus as the plasmid DNA, and moreover if the incubation time is too long, the ssRNA will have degraded by the time I detect it. My ssRNA has been labelled with Cy3 so that I can measure the flourescence of cells by flow cytometry after the incubation period.

My question is then: how much time should I incubate the cells after adding nanoparticles containing the ssRNA? I had thought about incubating the cells with the nanoparticles for 6h in Opti-MEM media and then replacing the Opti-MEM with regular MEM (as regular transfection protocols suggest), and then incubate cells overnight and submit them to flow cymetry first thing in the morning of the next day. Has anybody worked before with ssRNA? I haven't been able to find any paper discussing transfection of ssRNA...

Many thanks,

Adrian

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