Primescript rt enzyme mix 2

For the PCR detection of SVV RNA, total RNA from exosomes (RNase added to purified exosomes followed by incubation for 1 h at 37 °C before RNA extraction) and cells were extracted using a total exosome RNA and protein isolation kit (Life Technologies, USA) according to the manufacturer’s instructions. Total RNA from cell culture samples were isolated with the E.Z.N.A. total RNA kit I (Omega Bio-Tek) to quantify RNA copies of SVV in SVV-infected or exosome-treated cells. Detection of the number of copies of extracted RNA was performed using the Real-Time One-Step RT-PCR reagent (Takara). The reaction system was as follows: 2X One-Step RT-PCR Buffer III 10 μL, TaKaRa Ex Taq HS (5 U/μL) 0.4 μL, Prime Script RT Enzyme Mix II 0.4 μL, PCR forward primer (10 μM) 0.4 μL, PCR reverse primer (10 μM) 0.4 μL, SVV-3D probe 0.8 μL, total RNA 2 μL, and RNase-free dH₂O 5.2 μL (PCR primers and the SVV-3D probe were provided by our laboratory). The reaction times and temperatures of the PCR were 42 °C for 15 min (1 cycle) and 40 cycles of 94 °C for 10 s, 57 °C for 30 s, and 72 °C for 30 s. The Applied Biosystems 7300 Real-Time PCR System (Thermo Fisher) was used.

The primers and probes in the real-time qPCR assay were based on a previous report: forward primer, 5′-CCT CGGCGAGCGCTTTATCAC-3′; reverse primer, 5′-GGAAACT CATTCACCAAATCCTT-3′; probe, 5′-CGATGCAAGCTTTAT-3′ (Zsak et al., 2005 (link)). All primers and probes were synthesized by Sangon Biotech (Shanghai, China). The assay was performed in a LightCycler 480 II real-time fluorescent quantitative PCR instrument (Roche, Indianapolis, IN, United States) according to the a previously described method (Gallina et al., 2006 (link)). The reactions were conducted according to the manufacturer’s instructions in the one-step PrimeScript RT-PCR Kit (TaKaRa, Shiga, Japan). The total reaction in a 20-μl reaction volume contained 10 μl of 2 × one-step RT-PCR buffer III, 0.4 μl of TaKaRa Ex Taq HS, 0.4 μl of PrimeScript RT Enzyme Mix II, 0.4 μl of PCR forward primer, 0.4 μl of PCR reverse primer, 0.8 μl of probes, 2 μl of DNA template, and 5.6 μl RNase-free ddH₂O. Cycling proceeded at 42°C for 5 min and 95°C for 10 s, followed by 40 cycles of 95°C for 5 s and 60°C for 20 s. A melting curve analysis was performed using specific melting temperatures to verify the uniqueness of the amplified product. The data were analyzed using the LC480 System software.

The PCR assays were performed on the Rotor-Gene 3000 detection system (Corbett Research, Singapore) using One-Step PrimeScript™ RT-PCR Kit (Perfect Real Time) (Takara). All sets of reactions were carried out in a final volume of 20 l, each containing 10 l of 2× One-Step RT-PCR Buffer III, 0.4 l of MCMVf (10 M), 0.4 l of MCMVr (10 M), 0.8 l of probe (10 M), 0.4 l of Ex Taq™ (Takara) HS (5 U/l), 0.4 l of PrimeScript™ RT Enzyme Mix II, 1.0 l of total RNA or 1.0 l of RNA transcripts, and 6.6 l of RNase-free dH₂O. Amplification reactions were performed as follows: 42°C for 5 min; 95°C for 10 s; 40 cycles of 95°C for 5 s, and 60°C for 20 s. The specificity of this TaqMan assay was evaluated using six different reactions, including water control. Using the TaqMan probe, strong fluorescent signals were detected only from reactions with samples, while the signals from four other samples along with the water control were superposed to the baseline under optimized reaction conditions. These samples can be differentiated from the four other maize samples by comparing the signals of different levels. The PCR products were analyzed further by agarose gel electrophoresis. The assay with the sample displayed the expected band of 67 bp and an unexpected faint band above the 67-bp band, whereas those of the four others did not.

HAV (100 TCID₅₀) was mixed with serially diluted concentrations of
golvatinib or NAbs before the virus attached to cells (2 × 10⁵) and
then added to 2BS cells and incubated at 4°C for 1 h. The cells were washed
three times and total cellular RNA purified using RNeasy mini kit (Qiagen), as
described in the manufacturer’s instructions. Real-time quantitative PCR (qPCR)
was performed using One Step SYBR PrimeScript RT-PCR Kit (TaKaRa) in a MX3005p
RT-PCR instrument (Agilent). The 20-μL reaction contained 12.5 μL 2 × One Step
SYBR RT-PCR Buffer III, 0.5 μL TaKaRa Ex Taq HS, 0.5 μL PrimeScript RT Enzyme
Mix II, 0.5 μL each of 10 μM forward (5′-TGG AAT CAC ATT AAA GCA AGC AA-3′) and
reverse (5′-GGA ACA CGA AAT CTC AAA GTT GAC T-3′) primers, 2 μL total RNA, and 4
μL RNase-free H₂O. The thermal profile for qPCR was 42°C for 5 min
for reverse transcription, 95°C for 10 s for reverse transcription inactivation;
this was followed by 40 cycles of denaturation at 95°C for 10 s and annealing
and extension at 60°C for 30 s. GAPDH was used as the housekeeping gene to
normalize samples (forward 5′-CTG TTG CTG TAG CCA AAT TCGT-3′, reverse 5′-ACC
CAC TCC TCC ACC TTT GAC-3′). The analysis of relative levels of HAV RNA in
different samples was performed by comparative 2^−ΔΔCT method [58 (link)].

Total RNA was extracted from the grinding supernatant of Culicoides pools using RNAiso Plus (TaKaRa), following the manufacturer’s recommendations, and stored at −80°C. To detect BTV and AHSV in Culicoides pools, specific primer pairs and TaqMan probes for reverse transcription quantitative PCR (RT-qPCR) were employed (Hofmann et al., 2008 (link); Guthrie et al., 2013 (link)). The reaction mix comprised of 2 µL of RNA template, 10 µL of 2× One Step RT-PCR buffer III;, 0.4 µL of TaKaRa Ex Taq HS (5 U/μL), 0.4 µL of PrimeScript RT Enzyme Mix II, 0.4 µL of ROX Reference Dye II (50×), 0.4 µL of 10 µM of each primer and probe, and 5.6 µL of ddH₂O. The reactions were conducted using One Step PrimeScript™ RT-PCR Kit (TaKaRa) on a Fast 7500 Real-Time PCR machine (Applied Biosystems, Carlsbad, CA, USA) under the following conditions: 42°C for 5 min, 95°C for 10 s, and 35 cycles of 95°C for 3 s and 60°C for 30 s.