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
You may not have found relevant papers if you are using single-stranded RNA as keyword - the proper search term would be microRNA mimic.
However, microRNA mimics are in general double-stranded (see below paper for a comparison of ss and ds mimics). They are also generally chemically modified to increase nuclease resistance, you'll not see a good effect without such modification! You can easily buy such modified mimics from a number of companies... I do hope that you have considered the controls you are going to use in your experiments? MicroRNAs are notorious for potential off-target effects... please see this webinar for more detail
https://www.oligotherapeutics.org/guidelines-for-experiments-using-antisense-oligonucleotides-and-double-stranded-rnas/
In regards to the transfection - my own experience with single stranded fluorescent-mod antisense oligonucleotides in cell lines using lipid transfection is that you should start looking at transfection efficacy about 4 hours after the start of transfection! Definitly NOT 48 hours. Obviously your nanoparticles might take longer than that, but I'd certainly start at that timepoint. You can easily check location of your fluorescent mimic using an inverted fluorescent microcope. No need to remove cells from culture plates!
You may need to remove the transfection mixture to get good signal-to-noise ration, but you can always add it back on. Though doing a proper time-course study might make a nice supplementary figure in a future paper and certainly counts in any thesis...
Article Argonaute 2-dependent Regulation of Gene Expression by Singl...
5 min for equilibration and additional 20 min for formation of intact micelles
I used Lipofectamine 2000 for HepG2 and HEK293T cells. The 48 hour wait for transfection efficiency is if you are looking at fluoresent proteins. For fluorescently tagged oligos, 4 hours is about right.
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