Sensifast sybr fluorescein kit

After 168 h of culture, the cells were rinsed with HBSS and homogenized by TriReagent^®, Sigma Aldrich, Poznań, Poland. Total RNA was isolated using phenol–chloroform method as previously described by Chomczynski & Sacchi [56 (link)]. The RNA was diluted in DEPC-treated water and analyzed using a nano-spectrometer (WPA Biowave II, Biochrom, Cambourne, Cambridge, UK). Genomic DNA digestion and cDNA synthesis were performed using PrimeScript kit (Takara, Clontech, Mountain View, CA, USA). For each reaction, 150 ng of total RNA was used. Both processes were performed in accordance with the manufacturers’ instructions using a T100 Thermal Cycler (Bio-Rad, Hercules, CA, USA).
The qRT-PCR reactions were performed using a CFX ConnectTM Real-Time PCR Detection System (BioRad, Hercules, CA, USA). The reaction mixture contained 2 μL of cDNA in a total volume of 20 μL using SensiFast SYBR & Fluorescein Kit (Bioline, Cincinnati, OH, USA). Primer concentration in each reaction equaled 500 nM; primer sequences used in individual reactions are listed in Table 1. The average fold change in the gene expression of experimental cultures was compared with control cultures and calculated by the 2−DDCt method in relation to the housekeeping gene GAPDH.

C. difficile was cultivated in TY medium (pH 7.4) supplemented with 2 µg/ml thiamphenicol and 1 µg/ml nisin and harvested at T₃ (defined as three hours after the start of transition phase; OD₆₀₀ of 1.0 [approximately equivalent to H₈ on plates]). Aliquots (3 ml) of culture were immediately mixed with 3 ml of ice-cold ethanol-acetone (1:1) and stored at −80°C. RNA was purified and DNase I treated (Ambion) as previously described (29 (link), 35 (link), 77 (link)), and cDNA was synthesized using random hexamers (77 (link)). Quantitative reverse transcription-PCR (qRT-PCR) analysis, using 50 ng cDNA per reaction and the SensiFAST SYBR & Fluorescein kit (Bioline), was performed in technical triplicates on a Roche Lightcycler 96. cDNA synthesis reaction mixtures containing no reverse transcriptase were included as a negative control to ensure that no genomic DNA contamination was present. Results are presented as the means and standard errors of the means for three biological replicates. Statistical significance was determined using a one-way ANOVA, followed by Dunnett’s multiple-comparison test (GraphPad Prism v6.0), to compare transcript levels between the rstA mutant and each rstA overexpression strain.

On the seventh day of the experiment, cells were homogenized using 1 ml TRI Reagent. Procedure of homogenization was performed directly in the culture dish. Total RNA was isolated using the phenol–chloroform method as previously described by Chomczynski & Sacchi 35. Obtained samples were diluted in DEPC‐treated water. Both, quality and quantity of isolated total RNA were determined using nano‐spectrometer (WPA Biowave II, Hercules, California, USA). Genomic DNA digestion and cDNA synthesis were performed with PrimeScript kit (Takara, Clontech, Kusatsu, Shiga, Japan). For each reaction, 150 ng of total RNA was used. Both processes were performed in accordance with the manufacturers’ instructions using a T100 Thermal Cycler (Bio‐Rad, Hercules, California, USA).
The qRT‐PCR reactions were performed with a CFX ConnectTM Real‐Time PCR Detection System (BioRad). Reaction mixture contained 2 μl of cDNA in a total volume of 20 μl using SensiFast SYBR & Fluorescein Kit (Bioline, Cincinnati, Ohio, USA). The concentration of primers in each reaction equalled to 500 nM; primer sequences used in individual reactions are listed in Table 1. Relative gene expression analysis (Qn) was calculated in relation to the GAPDH housekeeping gene.
Moreover, we evaluated the ratio between BCL‐2 and BAX expression in each group by dividing Qn of BCL‐2 by Qn of BAX.