Rotor gene q machine

Cells were seeded in 6-well plates with 5 × 10⁵ cells per well and treated with MOD or cisplatin for 48 h. Total RNA was extracted using TRIzol reagent (Ambion, Carlsbad, CA, USA) and isopropanol precipitation. Complementary DNA (cDNA) synthesis, converting one μg of total RNA, was performed using oligo dT primer and RevertAid Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA). Template and solution including SYBR Green PCR Master Mix (Qiagen, Valencia, CA, USA) and primers were used for quantitative RT-PCR (qPCR) which was performed with a Rotor Gene Q machine (Qiagen, Hilden, Germany). Primers used were: Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward, 5′- GAAGGTGAAGGTCGGAGTCA-3′ and reverse, 5′-CATGGGTGGAATCATATTGGA-3′; KDM1B forward, 5′-GCGTGCTGATGTCTGTGATT-3′ and reverse, 5′-TTGTGGGATCTGGGACCTC-3′; DCLRE1B forward, 5′-CAACACCAATTGCAATCCAG-3′ and reverse, 5′-AGCCTTCTCCTCCACTGTGA-3′. qPCR protocol was 95 °C for 5 min, with 40 cycles at 95 °C for 5 s and 60 °C for 10 s. Each sample was analyzed in duplicate and expression values were normalized against GAPDH.

The Arabidopsis total RNA was extracted with TRIzol Reagent (CWBIO, Beijing, China). Small RNA was extracted using an EASYspin Plant microRNA Extract kit (RN40, Aidlab, Beijing, China). The DNA was removed by treating with RNase-free DNase I (RN34, Aidlab, China). After the detection of RNA samples quality by agarose electrophoresis, the same amount of RNA samples (1 μg) was used to generate cDNA with oligo dT primers and specific stem-loop reverse transcription primers of miRNA164 family members, respectively. The qRT-PCR was performed with SYBR Green Mix (CW0659, CWBIO, China). NCBI primer-blast online tools (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome) and qPrimerDB (https://biodb.swu.edu.cn/qprimerdb/) online database tools were used to aid in the design of specific primers. The reactions were incubated in a Rotor-Gene Q Machine (Qiagen, Hilden, Germany), AtActin2 and Histone H3 were used as internal controls for Arabidopsis and apple, respectively. Melting curve assay were performed to detection the specificity of qRT-PCR reactions. Gene amplification efficiencies and relative expression levels were analysed using the previous reported method [67 (link)]. All primers were listed in Table S3, see online supplementary material.

A SYBR green real-time RT-PCR assay specific for the Horsens strain was designed. The primers were designed to target ORF3, spanning the recombination breakpoint resulting in a 251 bp amplicon. The real-time RT-PCR was run on a Rotor-Gene Q machine (QIAGEN) in a total volume of 25 µL using the Qiagen OneStep RT-PCR kit with 2 µL extracted RNA, 1.5 µM of each primer and 2× SYBR green (SYBR Green I Nucleic Acid Gel Stain 10,000×, CAMBREX) final concentrations. The amplification profile was: 50 °C, 30 min; 95 °C, 15 min; 45 cycles (94 °C, 15 s; 60 °C, 15 s; 72 °C, 60 s); 72 °C, 5 min; 50 °C, 1 min, immediately followed by a melting point analysis, ramping from 50 to 99 °C, rising 1 °C per step, holding 5 s at each temperature increase. The fluorescence signal was measured at 72 °C during each PCR cycle and at each temperature increase during melting analysis. The fluorescent signals were analyzed with Rotor-Gene Q software v. 2.3.1 (QIAGEN), setting the NTC threshold at 10% and cycle threshold at 0.01.