Criterion tbe urea precast gels

Two micrograms of each RNA sample was loaded on 5% or 10% Criterion TBE-urea precast gels (Bio-Rad) and electrophoresed at 70 V. Next, the RNA samples were transferred to a Zeta-Probe GT membrane (Bio-Rad) using a Trans-Blot SD semidry transfer apparatus (Bio-rad) following manufacturer's guidelines. Transferred RNA was UV crosslinked and hybridized overnight with 100 ng/mL of 5′ biotinylated DNA probe (Supplemental Table S2) in ULTRAhyb (Ambion) hybridization buffer at 42°C. Blots were developed using a BrightStar BioDetect kit protocol (Ambion), imaged with a ChemiDoc MP imager (Bio-Rad) and quantified using Image Lab software version 5.2.1 (Bio-Rad). Signal intensity corresponding to each sRNA or mRNA was normalized to that of either ssrA or 5S rRNA, which served as internal loading controls. Decay curves corresponding to RNA stability time course experiments were generated by using GraphPad Prism version 5.0.

Two micrograms of each RNA sample was loaded on 10% polyacrylamide gels containing 7 M urea or loaded onto 10% Criterion TBE-urea precast gels (Bio-Rad) and electrophoresed at 85 V. RNA samples were transferred to a Zeta-Probe GT membrane (Bio-Rad) using a Trans-Blot SD semidry transfer apparatus (Bio-Rad) following the manufacturer’s guidelines. Transferred RNA was UV cross-linked and hybridized overnight with 100 ng/ml of 5′ biotinylated DNA probe (Table S3) in Ultrahyb (Ambion) hybridization buffer at 42°C. Blots were developed using a BrightStar BioDetect kit protocol (Ambion), imaged with a ChemiDoc MP imager (Bio-Rad), and quantified using Image Lab software version 5.2.1 (Bio-Rad). Signal intensity corresponding to each sRNA was normalized to that of 5S rRNA, which served as an internal loading control. Decay curves corresponding to RNA stability time course experiments were generated by using GraphPad Prism version 7.0.

Northern blotting was performed by following previous report,^[67] with significant modifications. Briefly, denatured 10–15 µg cellular total RNAs or 200–500 ng extracellular total RNAs were separated by 15% Criterion TBE‐Urea PreCast Gels (Bio‐Rad). The gels were stained with SYBR‐Gold (ThermoFisher Scientific) and transferred onto positively charged Nylon membrane (Sigma Aldrich) using Trans‐Blot Turbo Transfer System (Bio‐Rad). The membranes were then crosslinked with EDC at 60 ℃ for 1–2 h and prehybridized with ULTRAhyb Ultrasensitive Hybridization Buffer (Ambion). 50 pmol mL⁻¹ Biotin‐labeled Locked Nucleic Acid‐modified DNA probes (designed and synthesized by Qiagen) were used for hybridization at 37 ℃ overnight. After washing sequentially with high stringent buffer, low stringent buffer, and 1× SSC, the blots were then processed and developed using Chemiluminescent Nucleic Acid Detection Module Kit (ThermoFisher Scientific).