Loading dye

Semi-qRT-PCR was performed with Taq DNA polymerase (New England Biolabs). A PCR reaction (10 μl) comprised 10 × ThermoPol reaction buffer (1 μl), 10 mM dNTP (0.2 μl), 10 mM forward/reverse primers (0.25 μl each), Taq DNA polymerase (0.05 μl), diluted cDNA (1 μl), and autoclaved water (7.5 μl). PCR program was as follows: initial denaturation at 95°C for 3 min, followed by 25 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 68°C for 30 s, and a final extension at 68°C for 5 min, and products were held at 4–10°C. After that, 2 μl loading dye (Thermo Scientific) was added into PCR products, and then 10 μl products with loading dye were electrophoresed on 1.5% agarose gel with 1:1,000 GelRed (Biotium). Bands of cDNAs were visualized in GelDoc XR Imaging System (BIORAD), and band intensity was measured with Image J 1.45 s. The cDNA templates were adjusted to make sure that the band intensity of PCR products from different tissues were equalized before amplifying SlEF1a. After the equalization, the same amount of cDNA of different tissues was applied to amplify all the genes of interest. The gene specific primers are listed in Supplementary Table S1.

DNA protection activity of the studied extracts was analyzed using pUC19 plasmid DNA (pDNA). Plasmid isolation was performed by Thermo Scientific Genejet Plasmid Miniprep Kit. The reaction mixture contained 5 μL of Fenton's reagent (30 mM H₂O₂, 50 mM ascorbic acid, and 80 mM FeCI₃), 5 μL of these extracts at two different concentrations (5 and 10 mg/mL) and 3 μL of pDNA (300 μg/μL). Final volume of reaction mixture was brought up to 20 μL using double-distilled water. Positive control was composed of 12 μL of distilled water, 5 μL of Fenton's reagent and 3 μL of pDNA. Negative control involved only 17 μL of distilled water and 3 μL of pDNA. Samples were incubated for 30 min at 37°C and 4 μL loading dye (Thermo Scientific, USA) was added to the all mixtures. The DNA mixtures were run on 0.8% agarose gel and then visualized under ultraviolet light cabin. Biological replication of test was carried out at three times and band density was determined by the gel image analysis software (Quantum, Vision-Capt., Vilber Lourmat SAS, France) (Ozkan et al., 2015 (link)).

Peptide/miRNA complexes at N:P ratios of 1–15 were prepared. The PAGE gel was prepared by using components as mentioned in Table 2. Samples and 5 µL 20 bp DNA ladder (Takara, Kyoto, Japan) was added to the gel. The electrophoresis was run within tris-acetate buffer at 80 V for 2 h. Then the gel was stained in the TBE buffer with 2 µL loading dye (Thermo Fisher Scientific, Waltham, MA, USA). Images were taken using a ChemiDoc Touch (Bio-rad, Hercules, CA, USA) after staining the gel in Gel Red Nucleic Acid Gel Stain solution for 1 h.

Genomic DNA was extracted and purified form homogenized liver and pancreas tissues using Wizard^® Genomic DNA Purification kit (Promega, Madison, WI, USA). Extracted DNA was tested for concentration and purity by Nanodrop^® NA-1000 UV/Vis (ThermoFisher spectrophotometer, Wilmington, DE, USA). DNA fragmentation was assessed by horizontal electrophoresis using 2% agarose gel stained with ethidium bromide. A total of 10 μL of each DNA sample was mixed with 2 μL of the loading dye (ThermoFischer Scientific Inc, Waltham, MA, USA). Mixed samples were loaded to the prepared agarose gel. Electric current (90 volt) was applied for 45 min followed by visualization using UV trans-illuminator.

The sCA-miRNA pellet was dissolved in 100 μL of 0.02 M EDTA. The collected miRNA sample was mixed with loading dye (Thermo Fisher Scientific, Waltham, MA, USA) and loaded in a 4.5% NuSieve GTG agarose gel (Lonza, Basel, Switzerland). Imaging was performed using ChemiDoc Touch (Bio-rad, Hercules, CA, USA).