Rnapure bacteria kit dnase 1

Total RNA was isolated from P. aeruginosa strains without a chromosomal-fused lacZ (OD₆₀₀ = 2.5) cultured in LB medium at 37°C using an RNA pure Bacteria kit (DNase I) (CWBIO, Beijing, China). In accordance with the protocols, reverse transcription into complementary DNA was performed with RevertAid first-strand cDNA synthesis kits (Thermo Scientific). For qRT-PCR, reactions were 20 mL (10 mL SYBR Green Master Mix, 10 pM each primer, 10 ng final cDNA and specific volume of RNase-free water) for a ABI 7500 Real Time PCR machine (Applied Biosystems, Foster City, CA, USA). Each plate contained three technical replicates. RpoD was the internal control.

For qRT-PCR, A549 cells treated with different concentrations of EPS11 (0–9.00 nM) were centrifuged at 6000× g for 10 min, and total RNAs were extracted using the RNApure Bacteria Kit (DNase I) (CWBio, Beijing, China). Total RNAs were reverse transcribed into cDNA, and the transcriptional levels of different genes were determined by qRT-PCR with Sybr Green Premix Low rox (MDbio, Qingdao, China) and the QuantStudioTM 6 Flex (Thermo Fisher Scientific, Hudson, NH, USA). RNA integrity was assessed by RNA Nano6000 Assay Kit for the Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). Housekeeping gene β-actin was used as an internal reference. The relative gene expression was calculated using the 2^−ΔΔCt method with each transcript signal normalized to β-actin. Transcript signals for each treatment were compared to the transcript signals from the control group. Primers tubulin-f (5′-CTGCTCGCAGCTGGAGTGAG-3′) and tubulin-r (5′-CATAAATACTGCAGGAGGGC-3′) were used for amplification of βIII-tubulin encoding gene. Primers actin-f (5′-CACGATGGAGGGGCCGGACTCATC-3′) and actin-r (5′-TAAAGACCTCTATGCCAACACAGT-3′) were used for amplification of actin encoding gene. All qRT-PCR runs were conducted with three biological and three technical replicates.

To further determine the transcriptional level of target genes (ituD, ituA, ituB, ituC, degQ, degU, comA, glnR, sfp, codY, abrB and yczE), bacterial cultures were harvested after 24 h incubation. Total RNAs of these bacterial cultures were isolated using the commercial RNApure Bacteria kit (DNase I) (Cwbio, Beijing, China). Then, cDNA was synthesized using the extracted RNA samples and HiScript^® II Reverse Transcriptase SuperMix (Vazyme, Nanjing, China) according to the manufacturer’s instructions. Real-time PCR was carried out using FastStart Universal SYBR Green Master (Roche, Basel, Switzerland) on a StepOnePlus™ real-time PCR system (Applied Biosystems, Foster City, CA, USA). PCR conditions were as follows: pre-incubation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 20 s. The transcriptional levels of target genes were normalized against that of rspU [50 (link)].