Transscript uni one step gdna removal and cdna synthesis supermix kit

Real-time quantitative PCR (qRT-PCR) was performed using an Applied Biosystems 7500 real-time PCR instrument (Thermo Fisher, Carlsbad, CA, USA) and a two-step qPCR kit (PrimeScript™RT Reagent Kit and SYBR Premix ExTaq™, TaKaRa, Tokyo Metropolis, Japan). The PCR protocol was set as follows: 95°C for 7 min, followed by 39 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s. Total RNA was prepared from the freshly sampled potato pollens using the RNA simple Total RNA Kit (TIANGEN, Beijing, China). Then, cDNA was obtained via the reverse transcription of RNA samples using the TransScript^®-Uni One-Step gDNA Removal and cDNA Synthesis SuperMix kit (TransGen Biotech Co., Ltd, Beijing, China), following the manufacturer’s protocol. The StEF1α gene was assessed as the internal reference gene. The primers used for the PCR are listed in Supplementary Table S1. Relative gene expression levels were calculated using the 2^−ΔΔCt method (Schmittgen and Livak, 2008 (link)).

Salvia miltiorrhiza tissues were used for total RNA extraction using the Plant Total RNA Extraction kit (Aidlab, China) according to the user manual. Total RNA was transcribed into cDNA using the TransScript-Uni One-Step gDNA Removal and cDNA Synthesis SuperMix kit (TransGen Biotech). Reverse transcription of Smi-miR858 was carried out with specific stem-loop primers. RT–qPCR was performed using the TransStart Tip Green qPCR SuperMix (TransGen Biotech) and analyzed using the Bio-Rad CFX96 detection system. Three biological replicates and four technical replicates for each biological replicate were performed. S. miltiorrhiza 5.8S rRNA and SmUbiquitin were used as internal controls for the analysis of miR858s and protein-coding genes, respectively. The primers used for RT–PCR analysis are listed in Supplementary Data Table S2.

A total 200 μL supernatant was subjected to RNA and DNA extraction, according to the manufacturer’s instructions, using EasyPure Viral DNA/RNA Kit (TransGen Biotech, Beijing, China). Reverse transcription was performed using the TransScript-Uni One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (TransGen Biotech, Beijing, China). Bacterial DNA was extracted from culture broth using the bacterial genomic DNA kit. We used polymerase chain reaction (PCR) to detect common swine pathogens, including porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus 3 (PCV3), classic swine fever virus (CSFV), pseudorabies virus (PRV) and S. suis [42 (link),43 (link)]. The primers used for the detection of viral and bacterial genes are listed in Table S1. The genomes of PCV2-positive samples were amplified using two overlapping primer pairs (Table S1). The PCR procedure was as follows: 95 °C for 3 min, 35 cycles at 95 °C for 1 min, 56 °C for 1 min (different primer pairs choose different optimum temperatures), and 72 °C for 1 min, with a final extension at 72 °C for 10 min. The PCR product was sequenced by the Sanger method.