Total rna mini kit

The mRNA level of the ABCB1 gene was assessed using quantitative real-time PCR. Briefly, HL-60 cells were seeded in 6-well plates (4.0 × 10⁵ cells/mL) in 2 mL of growth medium and treated with 5a for 24 h. Total RNA was extracted using the Total RNA Mini Kit (A&A Biotechnology, Gdynia, Poland) according to the manufacturer’s protocol. The concentration of RNA used for further experiments was 500 ng. cDNA was synthesized using Transcriba Kit (A&A Biotechnology, Gdynia, Poland). The amplification of cDNA was performed using RT PCR Mix SYBR (A&A Biotechnology, Gdynia, Poland) and gene specific primers (Table 3) in the Stratagene Mx3005P QPCR System (Agilent Technologies, Inc. Santa Clara, CA, USA) according to the manufacturer’s guidelines. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a reference gene. The expression level of ABCB1 gene was determined by the 2^−∆∆CT method [23 (link)].

Total RNA was isolated from placentas using the Total RNA Mini Kit (A&A Biotechnology, Poland), according to the manufacturer’s protocols. The RNA quality and purity of the samples were determined spectrophotometrically (NanoDrop, Thermo Fisher Scientific, Poland). The samples were qualified for further analysis when the OD 260/280 ratio was between 1.8 and 2.0. The reverse transcription of 2000 ng of pure RNA into cDNA was conducted by the Maxima First Strand cDNA kit (Thermo Fisher Scientific, Poland) according to the manufacturer’s instructions. The obtained cDNA was stored at − 20 °C for further analysis [12 (link)].

Gene expression was initially examined in bacteria cultured for 20 h in iron/heme rich (Hm) or depleted (DIP) medium using microarray analysis as reported previously [13 (link),16 (link)]. Additionally, reverse transcriptase–quantitative polymerase chain reaction (RT-qPCR) was used as reported previously [16 (link),40 (link)]. Total RNA was isolated from 5 × 10⁷ to 4 × 10⁸P. gingivalis cells grown for 4, 10, and 24 h in iron/heme rich or iron/heme depleted medium, or one hour after exposure to oxidative stress using the Total RNA Mini Kit (A&A Biotechnology, Gdynia, Poland) and the Clean-Up RNA Concentrator Kit (A&A Biotechnology). Reverse transcription was carried out using total RNA and random hexamers with the SensiFAST cDNA Synthesis Kit (Bioline, London, UK). qPCR was performed using the SensiFAST SYBR No-ROX Kit (Bioline) and LightCycler 96 (Roche, Basel, Switzerland). All primers are listed in Table S1. Samples were analyzed in triplicate in two biological replicates and melting curves were generated to measure the quality of amplified products. Relative changes in gene expression were determined using LightCycler 96 software and P. gingivalis 16S rRNA (PGA7_00000960) as the reference gene.

Total RNA was isolated from the cells by utilizing a Total RNA mini kit (A&A Biotechnology, Gdynia, Poland). The RNA was then purified and stored at −80 °C, and reverse transcription (1 μg of total RNA) was performed using a High Capacity cDNA kit (Applied Biosystems, Foster City, CA, USA). The procedures were performed according to the manufacturer’s protocols.

The placental tissue samples stored at −20 °C were used for total RNA isolation using the Total RNA Mini Kit according to the manufacturer’s instructions (A&A biotechnology, Gdynia, Poland). The concentration and purity of the RNA was determined spectrophotomerically (NanoDrop, Thermo Fisher Scientific, Grand Island, NY, USA) and 2000 ng of pure RNA (OD_260/280 between 1.8–2.0) was transcribed into cDNA using a Maxima First Strand cDNA kit (Thermo Fisher Scientific, Waltham, MA, USA). The expression of the selected genes (Table 4) was analysed using commercial RT2 Profiler PCR Assays (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions in a LightCycler 480 (Roche, Mannheim, Germany) device.
The reactions were normalised according to GAPDH and YWHAZ as housekeeping genes, and to a reference sample including pooled cDNA from the study and control groups. Normalized gene expression was calculated as described by Pfaffl [62 (link)]. The results for relative gene expression were confirmed based on a measurement of absolute gene expression performed using a Droplet Digital^TM PCR System (Bio-Rad Laboratories, Hercules, CA, USA). The same RT2 Profiler PCR Assays from Qiagen were adopted for these analysis.