Nanodrop biophotometer plus

Each testis tissue was thawed, removed from RNAlater and pad-dried. Thereafter, RNA was extracted from each tissue using Innu Prep RNA mini kit (Analytik Jena, Jena, Germany), following the manufacturer’s protocol. The concentration and purity of extracted RNA was determined using a Nanodrop spectrophotometer (Eppendorf Nanodrop BioPhotometer plus, Hamburg, Germany. To determine RNA quality, gel electrophoresis using 1% agarose (w/v) in 1 × LB buffer, was performed and viewed using a UV transilluminator (ChemiDoc XRS, Bio-Rad Laboratories, Hercules, CA, USA). RNA samples which had OD_260/280 of 1.8–2.0 as determined by a Nanodrop spectrophotometer, and showed distinct 28S and 18S ribosomal RNA bands after gel electrophoresis, were selected for the downstream experiments.

Based on the maker’s instructions, total RNA was evaluated using Innu Prep RNA mini kit (Analytik Jena, Jena, Germany). Similarly, twenty mg of testis tissue was used to determine the purity and concentration of samples with the aid of spectrophotometer (Eppendorf Nanodrop BioPhotometer plus, Stevenage, United Kingdom). Samples with OD of 260/280 and 1.8–2.0 were used to know samples without impurities. Meanwhile, samples that were pure were exposed to gel electrophoresis with application of 1% agarose (w/v) in 1× LB buffer and observed with the aid of a UV transilluminator (ChemiDoc XRS, Bio-Rad Laboratories, Hercules, CA, USA), and pure samples were employed during the RT-qPCR amplification.

The bacterial isolates were cultured overnight at 37℃ in LB broth under antibiotic (meropenem) stress. Total RNA was extracted from the log-phase of two replicate cultures, using Trizol Reagent. Quantification and quality check of the extracted RNA was done using NanoDrop Biophotometer Plus (Eppendorf, Germany). Total RNA was digested with RNase-free DNaseI (New England Biolabs, USA) to remove any contaminating genomic DNA. Thereafter, cDNA was synthesized employing QuantiNova cDNA Synthesis Kit (Qiagen) and DNaseI-treated RNA as a template. To monitor gene expression levels, real-time RT-PCR using specific gene primers (as listed in Table S1) was carried out in Step One Plus Real-Time PCR System (Applied Biosystems). The 2^-ΔΔCT method (38 (link)) was used to calculate relative RNA expression levels, after normalizing with ribosomal housekeeping gene rpoB. All real-time RT-PCR experiments were repeated thrice, with rpoB gene as an internal control (39 (link)). Each target gene’s relative expression was then calibrated against the expression of an E. cloacae ATCC 13047 pan susceptible isolate (expression = 1). A twofold increase in gene expression in comparison to the reference strain was considered significant overexpression.