Gel doctm ez system

A target 17-mer JEV RNA sequence complementary to 3′ UTR stem-loop-targeted LNA gapmers was synthesized with HPLC grade by Ajinomoto Bio-Pharma Services GeneDesign, Inc. LNA gapmers (1.5 µM), synthesized JEV RNA (6 µM), and E. coli RNase H (50 units/mL; New England Biolabs, Ipswich, MA, USA) were mixed and incubated in 1 × RNase H reaction buffer at 37 °C for 120 min. The 20 µL reaction mixture was collected at 0, 5, 10, 30, 60, and 120 min after the reaction. The collected reaction mixture was immediately quenched by adding 1 µL of 0.5 M EDTA (NIPPON GENE, Tokyo, Japan) at pH 8.0 and denatured with an equal amount of 21 µL formamide (Wako). The samples were immediately used for a subsequent procedure or stored at −80 °C until use. They were mixed with an equal volume of Novex^TM TBE-Urea sample buffer (Thermo Fisher Scientific), heated at 70 °C for 4 min, and immediately placed on ice. Aliquots having a volume of 20 µL and 12 µL microRNA marker (New England Biolabs) were submitted to a Mini-PROTEAN 15% TBE-urea gel with ten wells (Bio-Rad, Santa Rosa, CA, USA) and electrophoresed at 200 V for 30 min. The gel was stained by SYBR® Green II (Thermo Fisher Scientific), and images were captured using the Gel Doc^TM EZ system (Bio-Rad). The band intensities were quantified using ImageJ software (version 1.53k; NIH).

A mixture of human Topo II (2 µl), substrate super coiled pHot1 DNA (0.25 µg), 50 µg/ml test compound (2 µl), and assay buffer (4 µl). The reaction started upon incubation of the mixture for 30 min at 37 °C. The reaction was terminated by the addition of proteinase K (50 µg/ml) and 10% sodium dodecylsulphate (2 µl) for 15 min at 37 °C. followed by incubation at 37 °C for 15 min. Then, the DNA was run for 1–2 h on 1% agarose gel in BioRad gel electrophoresis system followed by staining with GelRedTM stain for 2 h and destained for 15 min with TAE buffer. The gel was imaged via BioRad’s Gel DocTMEZ system. Both supercoiled and linear strands of DNA were incorporated into the gel as markers for DNA-Topo II intercalators. By using the GraphPad Prism version 5.0, the values of IC₅₀ were calculated. Each reaction was performed in duplicate, and at least three independent determinations of each IC₅₀ were made.
The data is available in a supplementary file.

A target 17-mer JEV RNA sequence complementary to 3′ UTR stem-loop-targeted LNA gapmers was synthesized with HPLC grade by Ajinomoto Bio-Pharma Services GeneDesign, Inc. LNA gapmers (1.5 µM), synthesized JEV RNA (6 µM), and E. coli RNase H (50 units/mL; New England Biolabs, Ipswich, MA, USA) were mixed and incubated in 1 × RNase H reaction buffer at 37 °C for 120 min. The 20 µL reaction mixture was collected at 0, 5, 10, 30, 60, and 120 min after the reaction. The collected reaction mixture was immediately quenched by adding 1 µL of 0.5 M EDTA (NIPPON GENE, Tokyo, Japan) at pH 8.0 and denatured with an equal amount of 21 µL formamide (Wako). The samples were immediately used for a subsequent procedure or stored at −80 °C until use. They were mixed with an equal volume of Novex^TM TBE-Urea sample buffer (Thermo Fisher Scientific), heated at 70 °C for 4 min, and immediately placed on ice. Aliquots having a volume of 20 µL and 12 µL microRNA marker (New England Biolabs) were submitted to a Mini-PROTEAN 15% TBE-urea gel with ten wells (Bio-Rad, Santa Rosa, CA, USA) and electrophoresed at 200 V for 30 min. The gel was stained by SYBR® Green II (Thermo Fisher Scientific), and images were captured using the Gel Doc^TM EZ system (Bio-Rad). The band intensities were quantified using ImageJ software (version 1.53k; NIH).