Direct zoltm rna miniprep plus kit

Total RNA was isolated from DIV 16 rat primary cultures using the Direct-zol TM RNA MiniPrep Plus kit (Zymo R2070) and reverse transcribed using the Quantiscript Reverse Transcriptase and random primers (Qiagen, 205311) following the manufacturers’ protocols. All rtPCR reactions were carried out using GoTaq Green PCR Master Mix (Promega, M7128) and a BioRad C1000 Touch thermocycler with the following conditions: 5 min at 94°C, followed by 40 cycles of 30 s at 94°C, 30 s at 60°C, and 45 s at 72°C, then a final 5 min at 72°C. The amplification products were visualized on 1% agarose gels under UV epifluorescence following ethidium bromide staining. Primers used include 5’-ATGACCGACTTCGAGAAGGACG-3’ (forward) and 5’-TCTGCGGCATCTGGTCAATGTG-3’ (reverse) for BAI1; 5’-ATGACCGACTTCGAGAAGGACG-3’ (forward) and 5’-CTGCACGTCATCAGCGGAAG-3’ (reverse) for BAI2; 5’-TAACCGGCCAGCAGTGTGAAG-3’ (forward) and 5’-CATTCCATCACCTGCCAGCAT C-3’ (reverse) for BAI3; and 5’-GATGATATCGCCGCGCTCGTC-3’ (forward) and 5’-AGCCAGGTCCAGACGCAGGAT-3’ (reverse) for actin. Reverse primers for BAI2 and BAI3 were designed using Snapgene.

For cells, total RNA was isolated from cultured keratinocytes using RNeasy Mini Kit (Qiagen, Valencia, CA, #74106) and treated with DNase I (Qiagen, #79254) to eliminate genomic DNA. For tissue, total RNA was isolated by following the manual of Direct-zol^TM RNA MiniPrep Plus kit (Zymo Research, R2073). The purity and concentration of RNAs were analyzed using a NanoDrop2000c (Thermo Fisher Scientific, ND-2000c). Following reverse transcriptase reactions using high-capacity RNA to cDNA kit (Life Technologies), qRT-PCR was performed to measure target genes using TaqMan probes and Fast reaction master mix reagents (Life Technologies). Relative expression of mRNAs was analyzed by the cycle of threshold (C_t) value of target genes and quantified by normalizing to ribosomal protein large P0 (RPLP0) for human keratinocytes and to β-Actin for mouse keratinocytes or tissues as housekeeping genes using the ΔΔC_t method^{48 (link)}.

Strains were cultured and treated with rifampicin as described for the chemical stability measurements. Samples (20 ml) were withdrawn and quenched with 4 ml of a solution of 5% phenol in ethanol. Cells were pelleted by centrifugation in an Eppendorf S‐4‐72 rotor at 4°C for 20 min. After suspension in 1.5 ml of Tri Reagent (MRC) using a MultiTherm agitator (37°C, 550 rpm, 10 min), cell debris was removed by centrifugation (20,000 g, 10 min, room temperature). An equal volume of ethanol was added to the supernatant and total RNA was prepared using a Direct‐zol^TM, RNA MiniPrep Plus kit (Zymo Research) following the manufacturer’s instructions. RNA was eluted in 80 µl of water. Concentration and purity were determined using a NanoDrop^TM spectrophotometer. RNA (2.5–4.0 µg) was heated at 65°C for 5 min in a 75 µl reaction containing 50% formamide, 2.5 M formaldehyde, 10 mM sodium MOPS, pH 7.0, 4 mM NaCl, 0.5 mM EDTA, 0.02% XC, 0.02% BPB and then vacuum blotted onto Hybond^TM‐XL filters (Amersham) using a P648 slot blot manifold (Amersham) following manufacturer’s instructions. After UV cross‐linking (Hoefer, UVC 500, 120,000 µJ/cm²), the blots were hybridized with 5′‐³²P‐DNA probes (Table S2). The rpsT, trxA and rpsO messages were hybridized with two probes to increase sensitivity. For normalization, the blots were stripped and then hybridized with a probe specific to 23S rRNA.

Cells of biofilm grown together with β-escin (5 μg mL⁻¹) at 37 °C for 24 h were scraped and washed in PBS as previously reported [37 (link)]. Total RNA was isolated using the Direct-zolTM RNA Miniprep Plus Kit (ZYMO RESEARCH, Irvine, CA, USA) according to the manufacturer’s instructions, and cDNA was obtained by reverse transcriptase (Bio-Rad, Milan, Italy) reaction using 1 μg of RNA. qRT-PCR was performed with 1 × SensiFAST TM SYBR Green master mix (total volume of 10 μL) (Meridiana Bioline) in an AriaMx Real-Time PCR instrument (Agilent Technologies, Inc., Milan, Italy) according to the manufacturer’s instructions. Fluorescence was measured using Agilent Aria 1.7 software (Agilent Technologies, Inc.). The expression of each gene was analyzed and normalized against the ACT1 gene using REST software (Relative Expression Software Tool, Weihenstephan, Germany, version 1.9.12) based on the Pfaffl method [38 (link),39 (link)]. The primer sequences used are listed in Table S1.

Cells of single or mixed biofilm grown together with myrtenol (12.5 μg mL⁻¹) at 37 °C for 24 h were scraped and washed in PBS as previously reported [28 (link)]. Total RNA was isolated using Direct-zolTM RNA Miniprep Plus Kit (ZYMO RESEARCH) according to the manufacturer’s instructions, and cDNA was obtained by reverse transcriptase (Bio-Rad, Milan, Italy) reaction using 1 μg of RNA. qRT-PCR was performed with 1 × SensiFASTTM SYBR Green master mix (total volume of 10 μL) (Meridiana Bioline) in an AriaMx Real-Time PCR instrument (Agilent Technologies, Inc., Milan Italy) according to the manufacturer’s instructions. Fluorescence was measured using Agilent Aria 1.7 software (Agilent Technologies, Inc.). The expression of each gene was analyzed and normalized against the ACT1 gene and 16SrRNA using REST software (Relative Expression Software Tool, Weihenstephan, Germany, version 1.9.12) based on the Pfaffl method [29 (link),30 (link)]. The primer sequences used are listed in Table 1.

Stored muscle tissues were retrieved and then homogenized using the Precellys Evolution homogenizer (Bertin Technologies, Rockville, MD, USA). Total RNA was lysed and isolated using the Direct-zol^TM RNA MiniPrep Plus Kit in which a genomic DNA elimination reagent (i.e., DNase I) is included to prevent DNA contamination (Zymo Research, Irvine, CA, USA). The quality of isolated RNA was assessed using the conventional A260/280 ratio and A260/230 ratio measurement (SpectraMax i3x; Molecular Devices, Sunnyvale, CA, USA). The total RNA (2 µg) was then reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s protocol. The cDNA samples were stored at −80 °C until analyzed.