Total rna mini plus

Total RNA from tachyzoites was extracted using Total RNA Mini Plus (A&A Biotechnology) according to the manufacturer’s instruction. The isolated RNA was concentrated using Clean-Up RNA Concentrator (A&A Biotechnology) and stored at -80°C. Single strand cDNA was synthesized using iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Inc.) and stored at -20°C.

RNA was isolated using Total RNA Mini Plus (A&A Biotechnology, Gdańsk, Poland). Complementary DNA (cDNA) was synthetized using qScript^TM cDNA Synthesis Kit (Quantabio, Beverly, MA, USA). SYBR Green real time—PCR was carried out according to the manufacturer’s instructions (Applied Biosystems, Waltham, MA, USA). qPCR signals (cT) in each experimental group were normalized to GAPDH levels.

Total RNA was isolated using Total RNA Mini Plus (A&A Biotechnology, Gdansk, Poland, 036–100) according to the manufacturer’s protocol. RNA (1μg) was reverse-transcribed into cDNA using oligo(dT)18 and TranScriba Kit (A&A Biotechnology, Gdansk Poland, 4000–100). qRT-PCR reactions were performed using the Sensitive RT HS-PCR Mix (A&A Biotechnology, Gdansk, Poland, 2017-2000P) and the real-time PCR thermal cycler (Bio-Rad). All TaqMan primers were from Thermo Fisher Scientific/Invitrogen: DYRK1A (Mm00432934_m1), GAPDH (cat. no. 4352932E). The relative levels of the transcripts were quantified by the 2−ΔΔCt method, using GAPDH as a reference gene.

Confluent monolayers of ICE-1 cells were stimulated over a period of 1 or 4 h by co-incubation of S. Enteritidis wild-type or S. Enteritidis fimH::kan mutant at a MOI of 100:1 bacteria to murine cells or with recombinant S. Enteritidis FimH protein (Grzymajlo et al., 2010 (link)) at a concentration of 40 μg/ml in a IEC medium or with IEC medium alone. Afterward, cells were washed three times with PBS, trypsinized, and total RNA was isolated using Total RNA Mini Plus (A&A Biotechnology, Gdańsk, Poland) according to the manufacturer’s instructions.
In the case of secreted interleukin analysis, confluent monolayers of ICE-1 cells were stimulated over a period of 6 h by co-incubation with S. Enteritidis or S. Enteritidis fimH::kan mutant at a MOI of 100:1 bacteria to murine cells, or with a PBS as control, in IEC medium without supplementation. After 6 h, media were collected, centrifuged to remove bacteria and frozen at -20^∘C. Secreted interleukins were determined by ELISA using Quantikine kits (R&D Systems, Minneapolis, MN, USA) or ELISA Ready-SET-Go (eBioscience, Inc., San Diego, CA, USA).

Total RNA was isolated for sequencing and real-time PCR using Total RNA Mini Plus (A&A Biotechnology, Gdańsk, Poland) according to the manufacturer’s recommendations. Total RNA concentration and purity were determined by measuring absorbance at 260/280 nm and 260/230 nm using a CLARIOstar Plus spectrophotometer (BMG LABTECH, Ortenberg, Germany).

Total RNA was isolated from the left ventricle using Total RNA Mini Plus (A&A Biotechnology, Gdynia, Poland) according to the manufacturer’s instructions. DNase-treated RNA (A&A Biotechnology, Gdynia, Poland) was reverse-transcribed using the RevertAid H Minus First Stand cDNA Synthesis Kit (Thermo Scientific, Pittsburgh, PA, USA). Real-time quantitative PCR was performed using the CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). SsoAdv Univer SYBR SMX (Bio-Rad, Hercules, CA, USA) was used to detect and quantify mRNA expression. The relative expression of each sample was determined after normalization to β-actin or 60S ribosomal protein L32 (RPL32) using the ΔΔCt method. A list of primers for real-time PCR is presented in Table 2.