Sybr green 2 rna gel stain

Aliquots of the ligation reaction were analyzed on 10% TBE-urea polyacrylamid gels (Thermo Fisher Scientific, EC6875BOX) to assess ligation efficiency. 2 µl ligation reaction or low range ssRNA ladder (New England Biolabs, N0364S) were heated for 5 min to 70 °C in 10 µl Novex TBE-urea sample buffer (Thermo Fisher Scientific, LC6876) and rapidly cooled before loading. Additionally, 1 µl GeneRuler Ultra Low Range DNA Ladder (Thermo Fisher Scientific, SM1213) was used for size estimation. Gels were stained 15 min at room temperature with SYBR Green II RNA gel stain (Thermo Fisher Scientific, S7564) and images acquired on a ChemiDoc Imaging System (Bio-Rad).

Polyacrylamide gel electrophoresis (PAGE) was used for the subsequent analysis of the isolated miRNAs. The aliquot of separated fractions was mixed 1:1 with a sample loading buffer (89 mM tris(hydroxymethyl)aminomethane, 89 mM boric acid, 2 mM EDTA, 7 M urea, 12% Ficoll, 0.01% xylene cyanole FF/bromphenol blue) and incubated for 4 min at 70 °C. All collected fractions were then loaded onto a 0.75 mm urea-polyacrylamide gel (20% gel with 8 M urea), and electrophoresis was performed with the Mini-Protean System (Bio-Rad, Hercules, CA, USA) at a constant voltage of 180 V in a Tris-Borate-EDTA running buffer. The gels were stained with SYBR Green II RNA Gel Stain (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Stained gels were analyzed using the ChemiDoc XRS + System (Bio-Rad, Hercules, CA, USA), with subsequent data processing in ImageLab software (Bio-Rad, Hercules, CA, USA).
Laemmli-SDS-PAGE electrophoresis with protein visualization by a silver staining method was used for the detection of protein residue in the samples of purified RNA [23 (link),24 (link)]. Stained gels were analyzed using the ChemiDoc XRS + System (Bio-Rad), with subsequent data processing in ImageLab software (Bio-Rad).

Collected samples (1.7 mL) were fixed with 0.2% glutaraldehyde (Sigma Aldrich) for 10 min and stored at −80 °C. Prior to counting, samples were fast-thawed at 26 °C, and bioaggregates were dismantled using 25 mM of EDTA and sonication. Samples were stained with 0.5 nM of SYBR® Green II RNA Gel Stain (Thermo Fisher Scientific S7564) in the dark for 15 min^{67 (link)}. Subsamples (150 µL) were analyzed with an Attune-Next acoustic focusing flow cytometer (Applied Biosystems) equipped with a syringe-based fluidic system at 408 and 488 nm wavelengths at a flow rate of 25 µL min^{−1 68 (link)}. Beads (nominal size 0.93 µm) (Polysciences) were used as a size standard.

Denaturing TBE-Urea 6% PAGE gels (Thermo Fisher # EC68652BOX) were run with 1× TBE-Urea sample buffer (Thermo Fisher # LC6876) and 1× TBE running buffer (Thermo Fisher # LC6675). Gels were prerun at 180 V (constant) for 20 min, then samples were loaded and run at 180 V (constant) for 3 h. Native TBE 6% PAGE gels (Thermo Fisher # EC62652BOX) were run with 1× Hi-Density TBE sample buffer (Thermo Fisher # LC6678) and 1× TBE running buffer. Gels were prerun at 180 V (constant) for 1 h, then samples were loaded and run at 180 V (constant) for 3 h. Total RNA was stained with 1× SYBR Green II RNA Gel Stain (Thermo Fisher # S7568) diluted in milliQ water for 10 min in the dark. G4s were selectively stained with 0.1 mg/ml NMM (Frontier Scientific # NMM58025MG) in milliQ water for 10 min in the dark. Stained gels were imaged on a Bio-Rad GelDoc Go Gel Imaging System using the SYBR Green setting. NMM stock was made at 10 mg/ml in dimethylformamide and stored in single use aliquots at −20 °C. For Native TBE PAGE, the mRNA was folded as described previously. Due to the different sensitivity, 100 ng and 2000 ng were loaded for staining with SYBR Green II and NMM, respectively.

Gel retardation assay was used to evaluate the dsRNA-p21 condensation ability of formulations. Samples containing 1 µg of dsRNA-p21 were mixed with the loading buffer (6×) and loaded in the well of 4% agarose gel in the presence of 1% SYBR Green II RNA gel stain (Molecular Probes, Eugene, OR). Samples were applied to 90 V electrodes in 0.5 × Tris-EDTA (TEA) (Millipore Sigma, Kenilworth, NJ) buffer for 20 min. RNA bands were tested with ChemiDoc XRS + electrophoretic imaging system (Bio-Rad Laboratories, Berkeley, CA, USA).