Total rna mini plus kit

The isolation of RNA from livers, kidneys, as well as hearts was performed using the Total RNA Mini Plus kit (Catalog Number 036–100, A&A Biotechnology, Gdynia, Poland). The RNA content was determined using a Multiscan Go spectrophotometer (Thermoscientific, Waltham, MA USA). RNA was reverse transcribed using the TranScriba cDNA Synthesis Kit for cDNA synthesis (Catalog Number 4000–100 A&A Biotechnology, Gdynia, Poland). As previously described, cDNA underwent real-time PCR analysis (CFX96 Touch™ Deep Well Real-Time PCR Detection System, Bio Rad, Hercules, CA, USA) in a mixture reaction containing the TaqMan Gene Expression Master mix (Cat. No. 4369016, Applied Biosystems, Foster City, CA, USA) and primers for the following genes: superoxide dismutase, heme oxygenase-1, and glutathione reductase (Invitrogen, Life Technologies, Oslo, Norway) [23 (link)]. Expression rates were determined as the difference in the normalized threshold cycle (CT) between the controls and the samples, with adjustment for amplification efficiency being made relative to the level of expression of the 18S housekeeping gene.

Total RNA was isolated from the overnight culture (control) and strains subjected to the ceviche preparation process using the Total RNA Mini Plus Kit (A&A Biotechnology, Gdynia, Poland) and purified and concentrated according to the manufacturer’s instructions using the CleanUp RNA Concentrator (A&A Biotechnology, Gdynia, Poland). RNA integrity of all samples was checked by loading 10 µL RNA into a 1.2% agarose gel in 0.5% TBE buffer and running at 90 V for 1 h. Once the RNA integrity is confirmed by visualizing the two bands (16S and 23S RNA) with little smearing. RNA concentration and purity were measured optically using a DeNovix DS11 FX spectrophotometer/fluorometer (DeNovix Inc., Wilmington, NC, USA) based on sample absorption at wavelengths of 260 nm and 280 nm. All RNA samples were immediately partially reverse-transcribed and the rest stored at −80 °C. All the RNA samples were normalized to 5 µg/µL and transcribed into the cDNA using the TranScriba kit (A&A Biotechnology, Gdynia, Poland) for the synthesis of first-strand cDNA. This kit uses recombinant MMLV reverse transcriptase, which has low RNAseH activity at 37–42 °C and optimal DNA polymerase activity. Template RNA was protected with a recombinant RNAse inhibitor. Random sequence hexamer was used as a primer.

The total RNA from L3 larvae was isolated with the Total RNA Mini Plus kit (036-100, A & A Biotechnology, Gdynia, Poland) according to the manufacturer’s instructions. cDNA was synthesized with 2 µg of RNA, oligo (dT) primers and reverse transcriptase from the TransScriba Kit (4000-100, A & A Biotechnology, Gdynia, Poland). The obtained cDNA was stored at −20 °C for further analysis.

The real time PCR analyses were performed as previously described [9 (link)]. Briefly, RNA was extracted using a Total RNA Mini Plus kit (A&A Biotechnology, Gdynia, Poland) according to the manufacturer’s protocol. The concentration and purity of isolated RNA were measured with an Epoch spectrophotometer (BioTek, Winooski, VT, USA). Two micrograms of total RNA were reverse transcribed into cDNA with RevertAid™ First Strand cDNA Synthesis kit according to the manufacturer’s instructions (Thermo Fisher Scientific Inc., USA.). The Real-Time PCR reactions were performed in duplicates with Real-Time HS 2× PCR Master Mix SYBR^® kit (A&A Biotechnology, Gdynia, Poland). The PCR conditions were: 95 °C for 10 min followed by 40 cycles of denaturation for 15 s at 95 °C, annealing for 15 s at 60 °C, extension for 15 s at 72 °C, and fluorescence reading for 10 s at 79 °C. Dynamic melting curve analysis was performed for all reactions. The data were collected using the StepOnePlus™ Real-Time PCR System (Life Technologies-Applied Biosystems, Grand Island, NY, USA). Primers were optimized for RT-PCR target gene expression, which was normalized against the housekeeping gene, RPL37. Statistically significant differences between treated and control cell lines were determined using comparative delta-delta Ct test. The primer sequences used in the study are given in Table 3 below.

The cDNA for human OPCs was acquired from ScienCell (1604), and human microglia total RNA was procured from Sti (37089RNA). RNA extraction was carried out using the fenozol-based Total RNA Mini Plus kit (A&A Biotechnology, 036–100,) as per the manufacturer’s instructions. The quantity and quality of mRNA were assessed through spectrophotometric analysis using a plate reader (Biotek, Synergy). RNA concentration was determined based on the optical density at 260 nm, and the samples were subsequently standardized. Subsequently, cDNA synthesis was performed using the TranScriba Kit (A&A Biotechnology, 4000–100,) following the manufacturer’s protocols. RT-qPCR was conducted using TaqMan Master Mix (Thermo Fisher, 4444556,) on the LightCycler480 (Roche, Switzerland). FAM dye-labeled TaqMan probes from Applied Biosystems (CA, United States) were employed. To calculate relative mRNA expression, the ΔΔCt method was utilized, with normalization to a reference gene based on absolute quantification.

The initial tumors and the cells cultured in different models for at least two or three passages were subjected to total RNA isolation using the Total RNA Mini Plus kit (A&A Biotechnology; Poland). The samples were treated with DNaseI (Sigma-Aldrich) and cDNA synthesis was performed with the SensiFAST™ cDNA Synthesis Kit according to the manufacturer’s instructions (Bioline, UK).

Expression of the CD59 cDNA in transfected cells was analyzed by reverse-transcription PCR (RT-PCR). Total RNA was isolated from porcine fetal fibroblasts using the Total RNA Mini Plus kit (A&A Biotechnology). The detection of human CD59 mRNA was carried out by reverse transcriptase reaction using the SuperScript^®VILO™ cDNA Synthesis Kit (Invitrogen). For PCR, 1 μl of cDNA solution was used. PCR was performed using 35 cycles (primer annealing 56 °C; extension 72 °C; denaturation 94 °C). The 327-bp cDNA fragment was amplified using primer F (5′-CTCGTCCTGGCTGTCTTCTG-3′) and primer R (5′-TGCTGCCAGAAATGGAGTCA-3′). RNA purity control was performed by the same set of primers. Simultaneously, to check the quality of the cDNA, the 319-bp cDNA fragment was amplified using primer β-actin F (5′-ACATCAAGGAGAAGCTGTGCTAC-3′) and primer β-actin R (5′-CTTCATGATGGAGTTGAAGGTAGTT-3′). The PCR products were separated on a 1.5 % agarose gel.