Applied biosystems 7500

Total DNA was extracted from mock (Agrobacterium-carrying empty vector)-, wt TYLCV- or TYLCV-C85S-infiltrated tomato or N. benthamiana leaves at different time points. RCR reaction mixes consisted of 6 μl of SYBR Green supermix (BIO-RAD, United States), 0.10 μl of each primer (10 pmol) and 1.5 μl of DNA sample (10 ng/μl) in a total volume of 12 μl.
PCR reactions were done in an Applied Biosystems 7500 (Life Technologies) real-time PCR detection system. SlActin or NbActin was used as an internal control for tomato or N. benthamiana, respectively. Data analysis was performed using Applied Biosystems 7500 software version 2.0.6.

Total RNA was extracted from clinical samples and cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. Reverse transcription of mRNA and miRNAs were performed using the PrimeScript RT Master Mix and SYBR PrimeScript miRNA RT-PCR Kits, respectively (Takara Biotechnology, Dalian, China). Quantitative real-time PCR was performed using SYBR Premix Ex Taq (Tli RNaseH Plus; Takara Biotechnology, Dalian, China) using the Applied Biosystems 7500 (Life Technologies); β-actin or U6 were used as internal controls to normalize the relative expression level of the target genes and miRNAs, respectively.

To demonstrate the parasite burden in mice, 30.0 mg of cardiac tissue obtained in Section 2.6 was lysed for genomic DNA extraction following the guidelines (OMEGA Bio-Tek, Norcross, Georgia, USA), and the DNA extracts were stored at −20°C until use. Absolute quantitative real-time PCR (qPCR) was carried out to investigate the 529-bp fragments in the extracts according to the published research (45 (link)). The plasmids containing the amplified 529-bp fragments were also constructed, and an online tool (http://cels.uri.edu/gsc/cndna.html) was employed to calculate the copy numbers of the plasmids. For qPCR amplification, 6.0 μl of 2× ChamQ Universal SYBR qPCR MasterMix (Vazyme, Nanjing, China), 0.24 μl of each primer, 1 μl of DNA extracts, and double-distilled water to make a final volume of 12.0 µl were involved in each reaction. Amplification for each reaction was performed on an Applied Biosystems 7500 (Life Technologies, Carlsbad, CA, USA) by 30-s incubation at 95°C, followed by 40 cycles of 10 s at 95°C and 30 s at 60°C. At the end of the reaction between 60°C and 95°C with an increment of 0.05°C/s, a melting curve analysis was performed. Before further analysis, the melting curve of each amplification was identified with one uniform peak as expected.

We used the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) to extract total RNAs. The cDNAs were synthesized with the PrimeScript reverse transcriptase reagent kit (TaKaRa, Dalian, China). Quantitative RT-PCR was performed on Applied Biosystems 7500 (Life Technologies, Carlsbad, CA, USA) with the THUNDERBIRD SYBR qPCR Mix (Toyobo, Osaka, Japan). The glyceraldehyde-3-phosphate dehydrogenase (Gapdh) gene was used as the reference gene for data normalization. A complete list of primer sequences is provided in Table S1.

Total RNA was purified from tumor tissues using TRIzol Reagent (Takara, Japan) and reversed-transcribed to cDNA with Prime Script TM RT Master Mix (Takara, Japan) according to manufacturer’s instructions. Real-time PCR was performed with Applied Biosystems 7500 (Life Technologies Corporations, Carlsbad, CA, USA) with initial denaturation at 95 °C for 30 s, and followed by 40 cycles of denaturation (95 °C, 5 s), annealing (55 °C, 30 s), and extension (72 °C, 30 s). Relative expression changes were calculated with ΔΔCt method. GAPDH served as the internal control. The primer sequences are as follows:
GAPDH: Forward: 5′-CCAGCCCAGCAAGGATACTG-3′
Reverse: 5′-GGTATTCGAGAGAAGGGAGGGC-3′
IFN-β: Forward: 5′-TCCGAGCAGAGATCTTCAGGAA-3′
Reverse: 5′-TGCAACCACCACTCATTCTGAG-3′
IFN-γ: Forward: 5′-AGCAACAGCAAGGCGAAAA-3′
Reverse: 5′-CTGGACCTGTGGGTTGTTGA-3′
IRF3: Forward: 5′-AACCGTGGACTTGCACATCT-3′
Reverse: 5′-GCCATGCTGTGTTTTGTCCC-3′