Stem loop rt primer

qRT-PCR was performed to validate the expressions of miRNAs together with their target genes. Total RNA was isolated at 20 dpi from N. benthamiana leaves either infected with TbCSV/TbCSB or not, using TRIzol reagent. For miRNAs, the cDNA was synthesized by RT using the PrimeScript RT reagent Kit with gDNA Eraser with a special stem-loop RT primer according to the manufacturer’s instructions (TaKaRa, Dalian, China). The cDNA products were used as templates for real-time PCR analyses. The primers used for qRT-PCR assays are listed in (Additional file 1 Table S1). In addition, we also used qRT-PCR to analyze the expression of target genes, and the primers are also listed in Table S1. Purified total RNA was used as a template and reverse-transcribed using the PrimeScript RT reagent Kit (TaKaRa, Dalian, China) to obtain cDNA. The reactions consisted of incubation in 96-well plates at 98 °C for 2 min, followed by 40 cycles of 98 °C for 10 s, 60 °C for 10 s, and 68 °C for 30 s. The expression level of the N. benthamiana Ubiquitin C gene (UBC) was used as the reference [38 , 39 (link)]. Three biological samples were used for each treatment and each biological sample had a further three technical replicates during qPCR. The qPCR results were calculated using the 2^-ΔΔCT method [38 ].

Total mRNA and miRNA from human leukemic cells and murine BM cells were extracted by Trizol reagent with minor modifications 30 (link). After extraction, RNA concentration and quality were determined by measuring the absorbance at 260/280 nm (DS-11 spectrophotometer, DeNovix, Wilmington, DE, USA). cDNA for qRT-PCR analysis was synthesized by using a Q5 real-time PCR system (Applied Biosystems, Carlsbad, CA, USA). miR-182 and U6 small nuclear RNA (snRNA) were reversely transcribed using Stem-loop RT primer with PrimeScript™ RT Master Mix (Takara Bio, Tokyo, Japan). U6 snRNA was used as an endogenous control for qPCR with miR-182. The special primers for miR-182 and U6 were provided by RIBOBIO company (Guangzhou, China). Human GAPDH and murine β-actin were used as endogenous controls for qRT-PCR of human and murine samples, respectively. SYBR Green dye (Takara) was used to determine the expression of mRNA and miRNA. Relative expression was calculated using the 2^-ΔΔCT method. All of the primer sequences were shown in Table S2.