Fast evagreen qpcr master mix

The total RNA was isolated from cells using the methods described above. The first-strand cDNA was synthesized using an Omniscript Reverse Transcription Kit (Qiagen, Netherlands) in a 20-μL reaction system containing 2 μg total RNA. The 1-μL aliquot of the first-strand cDNA was amplified using primers designed to investigate the expression of target genes by real-time PCR. The reaction mixtures included 10 μL of 2× Fast Evagreen qPCR Master Mix (Biotium, USA), 2 μL of 10× ROX (Biotium), gene-specific primers at a final concentration of 0.5 μM, and 1 μL of cDNA. Real-time PCR was performed in a StepOne real-time PCR system (ABI Applied Biosystems, USA). The thermal cycling program consisted of 2 min at 96°C, followed by 45 cycles of 15 s at 96°C and 1 min at 60°C. Data collection was performed during the 60°C extension step. To account for variability in the total RNA input, the expression of the target genes was normalized to that of the EF1α gene. In addition, a negative control without first-strand cDNA was prepared. The relative expression was calculated using the comparative 2^−ΔΔCt method.

RNA was isolated from the tissues and cell cultures using TRIzol (Life Technologies) according to the manufacturer’s instructions as in (Henriquez et al., 2013 (link); Bustos et al., 2017 (link); 2023 (link)). To obtain complementary DNA (cDNA), 400 ng of RNA was used. cDNA quantification was performed by qPCR using 3 μL of the cDNA mix, 6 μL Fast Evagreen qPCR Master Mix (Biotium, 31003), 2 μL of nuclease-free water, and 1 μL of 10 mM primers with the program recommended by the maker. The relative abundance was measured by the ddCt method using the GAPDH gene as a control. Transcript detection was performed with specific primers for messenger RNA (mRNA): KMT2C: Fw 5′ TGT​TCA​CAG​TGT​GGT​CAA​TGT​T 3′; Rv 5′ GAG​GGT​CTA​GGC​AGT​AGG​TAT​G 3′; GAPDH: Fw 5′ ATG​GTG​AAG​GTC​GGT​GTG​AA 3′; Rv 5′ CAT​TCT​CGG​CCT​TGA​CTG​TG 3′.

RNA was isolated with the ReliaPrep RNA Cell Miniprep kit (Promega) according to the manufacturer’s protocol. 250 ng RNA were reverse transcribed into cDNA with SuperScript III or IV (Thermo Fisher Scientific) and an Oligo(dT)₁₅ primer (Promega). qPCR was performed with the Fast EvaGreen qPCR Master Mix (Biotium) or PowerTrack™ SYBR Green Master Mix (Thermo Fisher Scientific) and measured on an ABI7300 Real-Time PCR system (Applied Biosystems) in duplicates in 96-well plates. Specific cDNA quantities were calculated based on the ∆∆C_t method and normalized to GAPDH as a housekeeping gene. The primers used are listed in Supplementary Table 6.

qPCR assays targeting genes for NTM, Pseudomonas, and total bacteria were performed by previously published methods (34 (link), 53 (link), 54 (link)) as detailed in Text S6. All DNA samples were processed in triplicate with negative controls and standards. Assays were conducted with 96-well polypropylene plates on a CFX96 real-time quantitative thermocycler (Bio-Rad, Hercules, CA). Each 10-μl reaction mixture contained 5 μl of Fast EvaGreen qPCR master mix (Biotium, Fremont, CA), 0.8 μl of each primer (0.4 μM; IDT, Coralville, IA), 2.4 μl of water, and 1 μl of template DNA (~0.2 ng ⋅ μl⁻¹). PCR conditions for NTM and total 16S rRNA gene assays consisted of 40 cycles, whereas the Pseudomonas assay was performed with 35 cycles. Melting curve analysis of the PCR products was conducted following each assay to confirm that the fluorescence signal originated from specific PCR products and not from primer dimers or other artifacts. For all qPCR assays, a linear relationship between the log of the standards’ DNA copy number and the calculated threshold cycle value across the specified concentration range was confirmed (R² value of >0.99 in all cases). Amplification efficiencies, calculated by the method described by Pfaffl (55 (link)), varied from 1.8 to 2.0 across all assays, and these values are consistent with those reported in other studies (54 (link), 56 (link)).