Rna stdsens analysis kit

C. albicans (5 × 10⁶ cells) was cultured in the presence or absence of 10 µL/mL of EO (direct contact or vapor) at 30 °C for 6 h. Positive control (the use of 5 µg/mL of AmB) was also included in the study. Total RNA was extracted from each sample as previously described [29 (link)]. The concentration, purity, and quality of the extracted RNA were determined using the Experion system and the RNA StdSens analysis kit according to the manufacturer’s instructions (Bio-Rad, Hercules, CA, USA). Appropriate RNAs were used to perform quantitative reverse transcription polymerase chain reaction (RT-PCR).

Fallopian tube samples were removed from liquid nitrogen storage and homogenized using a Tissue Lyser (Qiagen, Hilden, Germany). Total RNA was extracted using the RNeasy Plus micro Kit (Qiagen) according to the manufacturer’s protocol. RNA quality and quantity were evaluated based on spectrophotometric measurements (Nanodrop 1000, NanoDrop Technologies, USA) and possible RNA degradation was measured with an RNA StdSens analysis kit and Experion (Experion™ Electrophoresis Station, Bio-Rad laboratories, Hercules, CA).

C. albicans (10⁵ cells) was cultured in the presence or absence of CSC at various concentrations (30 and 50%) at 30°C for 24 h under agitation. At the end of this incubation period, the cultures were centrifuged 10 min at 13,000 rpm, the supernatants were discarded, and each pellet was suspended in 0.6 ml of lysis buffer (1 M glycerol, 0.1 M EDTA). Glass beads (0.425-0.6 mm in diameter; 0.2 ml) were then added to each suspended pellet prior to sonication (4 × 1 min, followed by 2 min of incubation in ice) by means of a MiniBead-beater (Biospec Products, Bartlesville, OK, USA). Following cell lysis, the total RNA was extracted from each sample by means of the Illustra RNAspin Mini kit (GE Health Care UK Limited, Buckingham, UK). The concentration, purity, and quality of the extracted RNA were determined using the Experion system and the RNA StdSens analysis kit according to instructions provided by the manufacturer (Bio-Rad, Hercules, CA, USA). Appropriate RNAs were used to perform quantitative RT-PCR.

Total RNA and a larger RNA fraction (excluding RNAs of less than 200 nucleotides in length) were isolated from the PANC-1 cells with the miRNeasy Mini Kit (QIAGEN Inc., Valencia, CA, USA) according to the manufacturer’s protocol. A smaller RNA fraction (enriched RNAs of less than 200 nucleotides in length) was isolated from the PANC-1 cells with the miRNeasy Mini Kit and RNeasy MinElute Cleanup Kit (QIAGEN) according to the manufacturer’s protocol. The purified RNA was analyzed with an Experion automated electrophoresis system using the RNA StdSens Analysis Kit (Bio-Rad Laboratories Inc., Hercules, CA, USA).

RNA isolation from combined leaf and stem tissues was carried out using a Spectrum Plant Total RNA Kit (Sigma Aldrich) with minor modifications to the manufacturer’s protocol. Optimization of the protocol included: (i) decreasing the amount of ground tissue used for extraction (50 mg instead of the proposed 100 mg); (ii) incubating samples for 5 min at 56 °C during lysis; and (iii) using 750 μL of binding solution. DNase digestion was incorporated during RNA isolation using an On-Column DNase I Digestion Set (Sigma Aldrich, St. Louis, MO, USA). RNA concentration and quality was measured using an Experion Automated Electrophoresis System with an RNA StdSens Analysis Kit (Bio-Rad, Hercules, CA, USA).
High-quality DNA was isolated using a DNeasy Plant Pro Kit (Qiagen, Hilden, Germany) without changes to the manufacturer’s protocol. DNA yield was estimated by Nanodrop (ThermoFisher, Waltham, MA, USA) and quality assessed by electrophoresis on a 2% agarose gel.