Oligo d t 18 primers

Total RNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The integrity of the RNA was checked electrophoretically, and complementary DNA (cDNA) was synthesized using 4 μg of RNA with the Oligo d(T)₁₈ primers and M-MLV reverse transcriptase (TaKaRa Bio, Tokyo, Japan), according to the manufacturer’s protocol.

Total RNA was extracted from tomato leaves using RNAiso Plus reagent (TaKaRa, Kusatsu, Shiga, Japan) following the manufacturer’s instructions. RNA concentration and purity were measured using a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit in an Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). The RNA samples were treated with DNase I (TaKaRa, Kusatsu, Shiga, Japan) and reverse transcribed into first-strand cDNAs using M-MLV reverse transcriptase (RNase H Minus) and oligo (dT)₁₈ primers (TaKaRa, Kusatsu, Shiga, Japan).

Cells and tissues were lysed using Trizol Reagent (Invitrogen, CA, USA) following the standard procedures. To detect mature miR-338-3p, specific TaqMan miRNA Assay Probes (Applied Biosystems, Foster City, CA) were constructed. U6 snRNA served as the endogenous control. We calculated the comparative 2^-∆∆Ct for relative quantification in which ΔΔC_T = (C_{T miR-338-3p} − C_{T U6})_tumor − (C_{T miR-338-3p} − C_{T U6})_control.
For quantification of PTP1B and GAPDH mRNA, the RNA was reversely transcribed into cDNA with oligo d(T)18 primers (TaKaRa). Then, the quantitative reverse-transcription PCR was conducted using SYBR Green dye (Invitrogen). The specific primer sequences of PTP1B and GAPDH were as follows: PTP1B (forward): 5′-TTCTGAGCTGGGCTTGTTGT-3′; PTP1B (reverse): 5′-TGCAGCTAAAATGCAAACCCAT-3′; β-actin (forward): 5′-GCACCACACCTTCTACAATG-3′; β-actin (reverse): 5′-TGCTTGCTGATCCACATCTG-3′; GAPDH (forward): 5′- CGAGCCACATCGCTCAGACA-3′; and GAPDH (reverse): 5′- GTGGTGAAGACGCCAGTGGA-3′. β-Actin or GAPDH served as the endogenous control. The PTP1B mRNA were relatively quantified with the 2^−ΔΔCt method similar to that described above.

Real-time PCR was performed in triplicate to determine the viral load in the heart, liver, spleen, intestine, lungs, and bronchus samples that had been collected, using the ABI Prism 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA) with SYBR green fluorescence detection. Total RNA samples were extracted from the aforementioned tissues using TRIzol (Gibco) reagent according to the manufacturer’s instructions. Total RNA was reverse transcribed into complementary DNA (cDNA) using Moloney murine leukemia virus (M-MLV), reverse transcriptase, and Oligo(dT)₁₈ Primers (Takara, Dalian, China). The cDNA was prepared for real-time qRT-PCR using a SYBR^® qPCR Mix Reagent Kit (Takara). Real-time quantification PCR was performed to determine the absolute copy numbers of CDV in each tissue. A standard curve was generated by plotting the threshold values against the serially diluted plasmid DNA encoding the CDV H protein fragment. The primer pair, H5 and H6, was based on inserted gene H. A negative control reaction, lacking template DNA, was also as performed with using the above protocol.

Total RNA was extracted from cells using a TRIzol Kit (Invitrogen, Carlsbad, CA, USA), and 1 μg was used for cDNA synthesis primed with Oligo(dT)18 primers (Takara, Dalian, China). Then, qPCRs were carried out according to the manufacturer’s instructions using SsoFast EvaGreen Supermix on a CFX96 Real-Time System (Bio-Rad Laboratories, Hercules, CA, USA); the oligonucleotide primers are shown in Table S2. Each qPCR reaction was repeated at least 3 times, and β-actin was used as the internal control, as previously described^{28 (link)}.

Total RNA was isolated from hemocytes of parasitized S. litura larvae 5 days post-parasitization using RNAiso Plus (TaKaRa, Dalian, China), according to manufacturer’s instructions, including DNase treatment. The concentration and purity of each RNA sample was determined by measuring OD at A260/A280 using NanoDrop 2000. Samples with an A260/A280 ratio >2.0) were used to synthesize cDNA using Oligo d (T) 18 primers following manufacturer’s instruction (TaKaRa, Dalian, China). All cDNA samples were stored at −80°C for preservation. qRT-PCR was performed using SYBR PCR Kit (Takara, Dalin, China) with the ABI 7500 system following the cycling parameters: 50°C, 2 min; 95°C, 10 mim; 95°C, 5 sec, 60°C, 34 sec, 40 cycles; 95°C, 15 sec; 60°C, 1 min; 95°C, 30 sec; 60°C, 15 sec. The 2-ΔΔCT method was used to get the relative mRNA levels [59] (link). 18S rDNA gene was used as the housekeeping genes for normalization. Three replications have been carried out for per sample.