I am trying to prove that some of the in vitro effects I observe following exosome treatment in target cells are miRNA dependent. In this phase I do not want to focus on a specific miRNA but just inhibit the overall response to miRNAs, to then check for an overall change in response to exosomes. I looked for specific references but it's been quite difficult. Would Ago2 KD by siRNA do the trick? should I knock it out altogether with CRISPR/Cas9? please provide reference if possible.
Thank you in advance!
Marco
I suspect either AGO2KD or AGO2KO should provide you with some global information. Other AGO proteins can function in miRNA-mediated gene silencing, which may need you to do double KD/KO. See "Kim YK, Kim B, Kim VN. Re-evaluation of the roles of DROSHA, Export in 5, and DICER in microRNA biogenesis. Proc Natl Acad Sci U S A 2016 Mar 29;113(13):E1881-9. PMID: 26976605".
What is the source of the miRNAs?
Specifically, are you are targeting exosome delivered miRNAs?
For the effect of delivered miRNAs, we used RNase treatment of the vesicles or pre-treatment of the target cells with actinomycin D to interrogate the global gene silencing effects.
See
Article Platelet microparticle delivered microRNA-Let-7a promotes th...
Hi,
You may be interested in the following paper: Hauptmann et al. PNAS 112(38):11841-5 (2015).
Maybe see: Eckenfelder A. eta al., Nucleic Acids Res. 2017 Apr 20;45(7):4158-4173. doi: 10.1093/nar/gkw1289.
Thanks for your suggestions! Chinedu Anthony Anene aren't miRNAs supposed to be RNAse A resistant? Did you use a different RNAse? Thanks again
Marco Ponzetti you are welcome.
RNase A can degraded miRNAs, which we collaborated by functional analysis.
A systematic analysis by Aryani and Denecke reported similar results (https://www.ncbi.nlm.nih.gov/pubmed/25899823). The attached figure from the paper suggests that the other RNases are less effective with RNase A causing more than 80% degradation.
Effective degradation depends on the cleavage site. RNase A cleaves single-stranded RNA at C and U residues, which is abundant in miRNAs.
Also, the intended use of the RNase treated vesicles favours RNase A. Specifically, inhibiting the RNase after treating your vesicles is critical to ensure it does not confound your co-culture experiments. RNase A has effective inhibitors suitable for cell culture.
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