M mlv reverse transcriptase

cDNA was generated by adding 4 μl of Random Hexamers (Promega) to 2 μg of RNA template in 28 μl of water. The primers were annealed to the template for 5 min at 70°C and then placed on ice. To 14 μl of primer-annealed template was added 5 μl of 5X M-MLV reverse transcriptase buffer, 1.25 μl of dNTPs, 1.25 ul of RNase inhibitor, and either 1.5 μl of M-MLV reverse transcriptase (Promega) or water. cDNA was generated at 37°C for 1 h and then diluted 1∶50 in DNase-/RNase-free water and stored at −20°C. qRT-PCR was performed in 12.5 μl final volume using SYBR Green Supermix, 16S RNA as a normalizing control, and 57°C as an annealing temperature. Primer sets used are in Table S1.

To detect the accumulation of the virus in the fruit, total RNA was extracted (as above) from different regions of tomato fruit showing different external coloration, and reverse-transcribed into cDNA using the M-MLV Reverse Transcriptase (Promega, USA) with a TRV-RNA2-specific primer, 5’-GGGCGTAATAACGCTTACGTAGGC-3’. The RNA2 cDNA of TRV was amplified with the RNA2-specific primers (GenBank accession number AF406991), 5’-CGGTCTAGAGGCACTCAACTTTATAAACC-3’ and 5’-CGGGGATCCCTTCAGTTTTCTGTCAAACC-3’. The RNA2 cDNA of the viral coat protein (CP) was amplified with the primers, 5’-CTGACTTGATGGACGATTCTT-3’ and 5’-TGTTCGCCTTGGTAGTAGTA-3’. To detect the silencing efficiency of specific genes, the isolated total RNA was reverse-transcribed into cDNA using the M-MLV Reverse Transcriptase (Promega, USA) with an oligo (dT)₁₈ primer. Real-time quantitative RT-PCR was performed to analyze gene expression patterns, as above. A pairwise Student’s t test was performed to determine whether the qRT-PCR results were statistically different between two samples (* = P < 0.05;** = P < 0.01). The primers used are listed in Additional file 8: Table S3. Each analysis was analyzed separately for three different fruits.

Total RNA was extracted from rice tissues using an RNA Extraction kit (MG Med, Seoul, Korea), according to the manufacturer’s instructions. For synthesis of first-strand cDNA, 2 μg of total RNA was used for reverse transcription (RT) in 20 μL volume with oligo(dT)₁₅ primer and M-MLV reverse transcriptase (Promega, Madison, WI, USA). For quantification of miR164b, stem-loop pulsed RT was conducted from 2 μg of total RNA in 20 μL volume using a miR164b-specific stem-loop primer and M-MLV reverse transcriptase (Promega) with the following conditions: 16 °C for 30 min followed by pulsed RT of 40 cycles at 16 °C for 2 min, 42 °C for 1 min, and 50 °C for 1 s, and then inactivation of reverse transcription at 70 °C for 5 min [57 (link)]. All RT products were diluted with 80 µL distilled water.
qPCR was performed with gene-specific primers and normalized to UBIQUITIN5 (UBQ5) (Os01g22490) or rice U6 snRNA (Table S1) according to the 2^−ΔΔCt method [58 (link)]. The 20 µL reaction mixture included 2 µL cDNA from RT or stem-loop pulsed RT, 1 µL 0.5 µM primer, and 10 μL 2X GoTaq master mix (Promega). qPCR amplifications were conducted with a LightCycler 480 (Roche, Basel, Switzerland) using the following program: 94 °C for 2 min followed by 40 cycles of 94 °C for 15 s and 60 °C for 1 min.

Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA). First strand cDNA was synthesized using M-MLV reverse transcriptase (Promega, Madison, WI, USA). PCR was performed for following rat target genes-thioredoxin interacting protein (TXNIP; Gene Bank Accession No. NM 001008767), insulin receptor substrate-2 (IRS-2; NM 001168633), immunoglobulin heavy chain binding protein (Bip, alternatively called GRP78; M14050), CCAAT/enhancer binding protein homologous protein (CHOP; U30186), glucagon-like peptide 1 receptor (GLP-1R; NM 012728) and beta actin (BC063166) in a Dyad Thermal Cycler (MJ Research, Watertown, MA). First strand cDNA was synthesized using M-MLV reverse transcriptase (Promega, Madison, WI, USA). Each set of primers was designed using Primer3 (http://bioinfo.ut.ee/primer3-0.4.0/primer3/). Primer sequences used, concentration of MgCl₂, cycle number and annealing temperature for each target gene are listed in S1 Table. Images of PCR products were captured using BioCapt v1.01 software and densitometric analysis was performed with Bio1D v1.01 software (Vilber-Loumat, Marne la Vallée, France).