Luna one step rt qpcr kit

TRIzol (Invitrogen) was used to isolate total RNA from infiltrated “spots” in N. benthamiana leaves. For Northern blotting, RNAs were separated by agarose gel electrophoresis and transferred to a charged nylon membrane by capillary action in 10× MOPS [3-(N-morpholino)propanesulfonic acid]. After UV cross-linking, membranes were hybridized with [α-³²P]dATP-labeled DNA probes (cocktail consisting of three 15-nt to 20-nt oligonucleotides). Reverse transcription-quantitative PCR (RT-qPCR) was first performed by first treating total RNA samples with RQ1 DNase (Promega). Next, 80-bp to 200-bp PCR fragments were amplified using a Luna one-step RT-qPCR kit (New England BioLabs). Reference genes included the p14 silencing suppressor (if infiltrated) or actin (ACT2). Relative fold changes were calculated using the threshold cycle (2^−ΔΔCT) method. All primers were designed using Primer3 online software (71 (link)).

Regular PCRs were done using Phusion High-Fidelity DNA polymerase (New England Biolabs, Ipswich, MA, USA) and a C1000 thermal cycler (BioRad, Hercules, CA, USA). Stepwise RT-PCRs were done using the SuperScriptIII First-Strand Synthesis System (Themo Fisher Scientific) and the oligo OK1-R for priming, followed by thermal amplification with Phusion High-Fidelity PCR kit (New England Biolabs). The PCR conditions using the oligos OK1-F and -R were: 98 °C 2 min/ 35× (98 °C, 30 s; 50 °C, 30 s; and 72 °C, 6 min)/ 72 °C, 10 min. When using primer pairs OK2-F and -R or OK3-F and -R, the elongation time was reduced to 1 min.
Single-step fluorescent qPCR was done with Luna one-step RT-qPCR kit (New England Biolabs) using low profile clear 0.2 mL PCR tubes and optical ultraclear flat caps (BioRad). The reactions were conducted in a CFX96 real-time PCR detection system (BioRad) with CFX Manager 3. Cqs were auto calculated by the software, and ranged from ~190 to ~250. The single-step RT-qPCR conditions were as follows: 55 °C, 10 min/35× (95 °C, 10 s and 60 °C, 30 s, plate read).

Following two
rounds of d-sorbitol treatment, RNA was isolated from synchronized
trophozoite-stage
parasites (24 h post-treatment) using a RNeasy mini kit (Qiagen),
according to manufacturers’ instructions. Quantitative-RT PCR
was performed using the Luna One Step RT-qPCR kit (NEB) on an Agilent
MX3005P system. mRNA levels were normalized to the reference gene,
β actin (PF3D7_1246200), and expression levels compared using
the ΔΔC_t method. Primers used are detailed
in Table S1.