Random hexamers

First-strand cDNA was obtained by using the First Strand cDNA Synthesis Kit (GE Healthcare), with random hexamers, (GE Healthcare) according to the manufacturer’s protocol. RT products were stored at −80°C.

Total RNA was isolated using the highly efficient genomic DNA removal RNeasy plus kit (#74136, Qiagen, Valencia, CA, USA), according to the manufacturer’s protocol. The NanoDrop ND1000 spectrophotometer (Nyxor Biotech, Paris, France) was used in determining total RNA concentration. Reverse transcription of 1 μg total RNA with 2 μg random hexamers (GE Healthcare, Amersham, Buckinghamshire, UK) and Invitrogen™ SuperScript™ III reverse transcriptase (#18080-044; Thermo Fisher Scientific Inc., Grand Island, NY, USA) according to manufacturer’s instructions. The following primers were used in this study: MALAT1 forward 5′-AAAGCAAGGTCTCCCCACAAG-3′, reverse 5′-GGTCTGTGCTAGATCAAAAGGC-3′; c-Myc forward 5′-GGCTCCTGGCAAAAGGTCA-3′, reverse 5′-CTGCGTAGTTGTGCTGATGT-3′; cyclin D1 forward 5′-GCTGCGAAGTGGAAACCATC-3′, reverse 5′-CCTCCTTCTGCACACATTTGAA-3′; Axin2 forward 5′-ACAACAGCATTGTCTCCAAGCAGC-3′, reverse 5′-GCGCCTGGTCAAACATGATGGAAT-3′; LEF1 forward 5′-AGAACACCCCGATGACGGA-3′, reverse 5′-GGCATCATTATGTACCCGGAAT-3′; DKK1 forward 5′-CCTTGGATGGGTATTCCAGA-3′, reverse 5′-CCTGAGGCACAGTCTGATGA-3′; GAPDH (internal control) forward 5′-ATCATCCCTGCCTCTACTGG-3′, reverse 5′-GTCAGGTCCACCACTGACAC-3′. The data were displayed as 2^−ΔΔCt values and are representative of at least three independent experiments.

Viral RNA was extracted using the QIAxtractor (Qiagen, Hilden, Germany) platform. Extracted RNA was denatured at 97°C for 5 minutes and reverse transcription-PCR was performed using M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA, USA) with random hexamers (Pharmacia Biotech, Uppsala, Norway) by following a single thermal cycle of 25°C for 5 minutes, 37°C for 60 minutes and 95°C for 5 minutes [24 (link)].
5μl of the cDNA was used for G-typing and P-typing PCR reactions of each specimen, following previously established procedures for eight G (1–4, 8–10, 12) types and six P (4, 6, 8–11) types rotavirus strains [25 (link)–26 (link)].

cDNA was prepared by reverse transcription using total RNA as the template, and 1 μg of each RNA sample heat-treated at 65 °C was mixed with 20% five First Strand Buffer (Thermo Fisher Scientific), 1 mM dithiothreitol (Life Technologies, Gaithersburg, MD, USA), and 1 μg of each RNA sample heat-treated at 65 °C. The final concentration shown below was added to the sample: (Gaithersburg, MD, USA), 1.1-U/µL ribonuclease inhibitor (Takara Bio, Shiga, Japan), 0.5-mM dNTP mixture (Takara Bio), 5-U/µL Moloney-Mouse leukemia virus reverse transcriptase (Life technologies), and 55 ng/µL random Hexamers (Pharmacia Biotech, Milwaukee, WI, USA). The reaction solution was stored at 37 °C for 60 min and subsequently treated at 99 °C for 5 min to inactivate the residual enzymes for cDNA preparation.

DNA was isolated from leaves according to Doyle and Doyle [58 ]. RNA was isolated from leaves and stems using the Total RNA Plant Isolation kit (Sigma) according to the supplier’s instructions, after which a further DNase treatment was added. The quality and quantity of RNA were verified by agarose gel electrophoresis and spectrophotometric analysis. The absence of DNA from the RNA samples was tested by the null PCR amplification of the universal rDNA primer pair ITS1/ITS4, as described in Paolocci et al. [59 (link)]. The same primer pair was also used to prove the hybrid status of F1 plants by amplifying the genomic DNA from parental and hybrid lines and sequencing the resulting amplicons. Then cDNA from parental, hybrid F1 and F2 plants was synthesized from 3 μg of total RNA, using SuperScript III H-Reverse Transcriptase (Invitrogen) and 100 pmol of random hexamers (Pharmacia Biotech) according to supplier’s instructions.