Gel doc image analysis system

Using Zetasizer Nano ZS (Malvern) and Zetasizer Nano software, we could determine each molecular weight of Dsi RNA transcripts, as a result of a Debye plot calculation. We measured the intensities of scattered light at a single angle, while serially diluting the samples with nuclease-free water. Dsi RNA transcripts had 1.72 × 10⁴ ± 358 kDa and therefore, the molecular weight of Dsi RNPs (1:0.3, w/w) was estimated as 2.24 × 10⁴ ± 698 kDa. The hydrodynamic diameter and zeta potential of RNA particles were also measured by Zetasizer Nano ZS. All samples were filtered by syringe filter (0.22 μm MWCO) before measurement and the data was collected at a laser wavelength of 633 nm with a scattering angle of 173°.
For the stability studies of RNA particles against serum nucleases, we added fetal bovine serum solution (30% at the final concentration) to Dsi RNA transcripts (10 μg/mL) or Dsi RNPs solution (10 μg/mL), and incubated the mixture according to the indicated time period. We electrophoresed each mixture on 15% non-denaturing TBE gel and quantitatively analyzed the band intensities by using a Gel Doc image analysis system (Bio-Rad). Other additional tools or methods for characterization of RNA NPs are described in the Supplementary Information.

The BCA kit (Pierceprotein Stint® Inc., BCA Protein Assay Kit, Thermo Scientific USA) was used, a total 50 μg protein of SDS-PAGE was separated through electrophoresis after, and the nitrocellulose membrane (Hybond ECL, GE Healthcare Life sciences, UK) was transferred. The protein is a membrane to transfer 3% skim milk (BD Difco, USA) after each block with diluted primary antibodies phospho-ERK (1 : 1000; Cell Signaling, Danvers, MA, USA), ERK (1 : 2,000, Cell Signaling, Danvers, MA, USA), phospho-JNK (1 : 500; Santa Cruz, CA), JNK (1 : 500; Santa Cruz, CA), p65 (1 : 1,000; Cell Signaling, Danvers, MA, USA), NFAT1 (information), and β-actin (1 : 10000; Sigma-Aldrich, USA) antibody overnight. Tris-buffered saline with Tween 20 (+0.1% Tween 20; TBS-T) as a cleanser and secondary antibodies ((p)-ERK, p38, (p)-(p)-p65, HRP-anti-rabbit; p-JNK, HRP-anti-goat; and β-actin, JNK, HRP-anti-mouse) triggered the reaction. ECL Western blotting and the detection solution (Amersham detection reagents; GE Healthcare Life Sciences, UK) react with the Gel Doc image analysis system (Bio-Rad, Hercules) which was confirmed using protein bands.

RNA transcripts, RNAtr/DNA–Chol hybrids and RNAtr/DNA–Chol/FA–DNA hybrids at the indicated weight ratio were analysed on 3% agarose gel. To distinguish FA–DNA conjugates from DNA–Chol conjugates, we used the FAM-labelled FA–DNA conjugates (FA–DNA–FAM). Nucleotides bands were visible under ultraviolet irradiation after SYBR gold staining. Throughout the current studies, the gel images were obtained by a Gel Doc image analysis system (Bio-Rad, USA). To measure the FAM fluorescence bands, we used a 12 bit charge-coupled device camera (ISM 2000MM, Kodak, USA).