Forget me not evagreen qpcr master mix

To increase the annealing temperature, a sequence of AGCC was added to the first 17 nt of all mature miRNAs, and used as miRNA qPCR forward primers. The oligonucleotide 5′-GTG CAG GGT CCG AGG TAT TC-3′, which is derived from the hairpin or stem–loop structure, was used as a common miRNA qPCR reverse primer. Primers for the reference transcript human 5S ribosomal RNA were designed using the Primer3Plus program. The touchdown quantitative real-time PCR (TqPCR) reactions were set up by using the 2× Forget-Me-Not™ EvaGreen qPCR Master Mix (Biotium, Fremont, CA), and carried out by using CFX-Connect (Bio-Rad) as previously described (25–28 (link)). The TqPCR cycling program was as follows: 95°C for 3 min for 1 cycle; 95°C for 20 min and 66°C for 10 min, for 4 cycles by decreasing 3°C per cycle; and 95°C for 20 min, 55°C for 10 min and 70°C for 1 min, followed by plate read, for 40 cycles.
Five-fold serial dilutions were performed to determine the amplification efficiency for each qPCR primer pair. No template control was used as a negative control. All reactions were done in triplicate. To quantitatively assess the quantification cycle (Cq) deviation from the MsHP group, ΔCq values were calculated for the UHP groups by subtracting individual average Cq value from respective Cq value for the MsHP group: ΔCq = average Cq (MsHP) − average Cq (UHP).

After confirming primer specificity by amplicon sequencing, qPCR reactions were optimized using a Bio‐Rad CFX96 qPCR Instrument (Bio‐Rad Laboratories). The primer performance was initially evaluated using a gradient of melting temperatures (T_m) in a reaction volume of 10 µl containing 200 nM of each primer, 2 µl of DNA (80 ng DNA/2 µl) for the samples, or 2 µl of serial dilutions (corresponding to 10²–10⁷ template copies in the reaction) for the standard curve, 5 µl of 2× Forget‐Me‐Not™ EvaGreen® qPCR Master Mix (Biotium) and 2.4 µl of sterile nuclease‐free water. Thermocycling conditions were 95°C for 2 min, followed by 40 cycles of 95°C for 5 s, the optimized melting temperature for each primer pair for 10 s (between 58 and 61°C) and 72°C for 10 s. A final melting curve was generated by increasing the temperature up to 95°C in increments of 0.5°C every 10 s to confirm single reaction products were obtained. Control reactions included substitution of genomic DNA by water to confirm the absence of contamination.

RNA extractions from fruits of mock control, EV, pK7GWIWG2(i)-WRKY29-RNAi, or pK7GWIWG2(i)-WRKY64-RNAi were conducted using the Spectrum™ Plant Total RNA Kit (Sigma–Aldrich, MO, USA) five days after Agrobacterium infiltration. After DNaseI treatment, the cDNA synthesis was performed using LunaScript® RT SuperMix Kit (New England Biolabs, MA, USA). The quantitative real-time PCR was performed using the Forget-Me-Not™ EvaGreen qPCR Master Mix (Biotium, CA, USA) with an internal reference gene of FaGAPDH2 using LightCycler® 480 system (Roche, Basel, Switzerland). The primer sequences used this gene expression assay are FaWRKY29_F (5’-GGAGATCATTGAAGGGATGGAG-3’), FaWRKY29_R (5’-GAGGTCAATATCCTCTGCACTAAA-3’), FaWRKY64_F (5’-CACCTTCGCTAAATGGGAGT-3’), FaWRKY64_R (5’-TTACTTGTTTGGTCCACCGT-3’), FaGAPDH2_F (5’-CCCAAGTAAGGATGCCCCCATGTTCG-3’), and FaGAPDH2_R (5’-TTGGCAAGGGGAGCAAGACAGTTGGTAG-3’). Three biological and technical replications were used. The relative gene expression levels were calculated using the 2^−ΔΔCT.