Stepone real time pcr system software

Gene amplification was carried out using real-time polymerase chain reaction (qrt-PCR) with StepOne Real-Time PCR System software (Applied Biosystems) and the SYBR Green® PCR Master Mix commercial kit (Applied Biosystems). Three replicates were obtained for each experimental group, and each sample was individually amplified in triplicate. In each well, 10 µl of the reaction consisted of 8 µl of SYBR Green® PCR MasterMix (Applied Biosystems), with 0.2 mM of each primer (fowad and reverse), and 2 µl of the cDNA sample diluted at a 1:4 ratio. The thermocycling conditions were 95 °C for 10 min, followed by 45 cycles of 95 °C for 15 s and 60 °C for 1 min. After each PCR run, denaturation curve analysis was performed on each sample to detect oligonucleotide dimers and nonspecific products. To assess the amplification efficiency of each transcript, a standard curve was generated by serial dilution of cDNA. The GAPDH gene was amplified as an endogenous control. Gene expression analysis was performed based on the calculation of:
The characteristics of the selected genes for analysis are described in Table 1.

To assess TT12A-specific phage mobilization upon MMC-induction, LB and LB + MMC subcultures were grown for 6 hrs at 37 °C with shaking (220 rpm) and then centrifuged at 5000× g for 10 min. Supernatants were filtered through low-protein-binding 0.22 μm pore size membrane filters (Millex-GP; Merck Millipore Ltd., Burlington, MA, USA) and treated with DNase I (Invitrogen, Waltham, MA, USA) for 15 min to remove bacterial gDNA. Phage DNA was extracted from the lysate using the QIAamp DNA Mini Kit (Qiagen Inc., Valencia, CA, USA), and eluted with 50 μL nuclease-free water. Phage mobilization was determined by qPCR targeting the phage-borne toxin genes stx1 and stx2 as well as ΦPP10-carried nleL gene on the StepOne Real-Time PCR System software (v 2.3) (Applied Biosystems, Foster City, CA, USA). Statistical significance was determined using Prism (v.9.5.0) (GraphPad Software, San Diego, CA, USA) with two-way ANOVA with Sidak’s multiple comparisons test to compare non-induced to MMC-induced conditions.

The expression of PgE17 and PgGa1 was analyzed in leaf, stem, and root samples, as well as in fruit in the green, breaker, ripe, and overripe stages of ripening. The ribosomal 25S sub-unit gene from Nicotiana tabacum leaf was used as a reference gene to measure the relative expression as described in other works [20 (link), 21 (link)]. The green ripening state results were used for data normalization.
The qPCR primers for PgE17 were forward 5′-GGTACGGTCGTGCTGGTC-3′, reverse 5′-GGCGAAGATCCAGTGCTC-3′, and for PgGa1: forward 5′-AAAGGTTCACGCAGAAGATAGT-3′ and reverse 5’-CTGGTCCAAATTCGTTCTCTAT-3′. The qPCR reactions were performed using the Applied Biosystems™ Power SYBR™ Green PCR Master Mix kit, in a 96-well StepOne™ Real-Time PCR System (Applied Biosystems) thermocycler. Each reaction was assembled as follows: 10 μl of Power SYBR™ Green PCR Master Mix (2X), 200 ng of cDNA, and 100 nM of forward and reverse primers. The final volume was adjusted to 20 μl using Nuclease-Free Water. The PCR conditions were as follows, initial denaturation at 95 °C for 2 min, followed by 40 cycles of 5 s denaturation at 95 °C, an annealing and extension cycle at 52 °C for 30 s. Gathered data was analyzed using the StepOne™ Real-Time PCR System Software (Applied Biosystems). The relative fold changes in gene expression were calculated by the 2^−∆∆ct model.