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Nuclease stability assay

CG C. M. A. Gangemi
SA S. Alaimo
AP A. Pulvirenti
SG Sara García-Viñuales
DM D. Milardi
AF A. P. Falanga
MF M. E. Fragalà
GO G. Oliviero
GP G. Piccialli
NB N. Borbone
AF A. Ferro
AD A. D’Urso
CC C. M. Croce
RP R. Purrello
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Nuclease stability assay was performed in 10% Fetal Bovine Serum (FBS) (Sigma) in Dulbecco’s Modified Eagle Medium (DMEM) (Microgem) without phenol red at 37 °C. We used FBS instead of pure nucleases because the former mimics better the physiological conditions in which miRNAs operate. Indeed FBS contains several endo- and exo-nucleases and not a single nuclease47–49. For preparation of pre-treated or control samples, 3.5 nmol of oligonucleotide (ON) were dissolved in 125 µL of FBS or proper buffer, respectively. After 72 h of incubation samples were stored at −80 °C for 5 h, then lyophilized and re-dissolved in 10 µL Milli-Q water and 10 µL of loading buffer (glycerol/TBE 1× –30 mM KCl 1:9). 10 µL of the mixture was used for non-denaturing polyacrylamide gel electrophoresis (PAGE).

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