Onestep rt pcr reaction kit

Viral RNA was extracted from 140 µL of the blood samples using the QIAamp viral RNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. A diagnostic assay developed by Panning et al.,^{11 (link)} for filovirus species and carried out with a OneStep RT-PCR reaction kit (Qiagen, Hilden, Germany) was used in a 25 µL total reaction volume, including 3 µL of the extracted RNA. The real-time diagnostic assay used five optimised large (L)-gene primers and three probes, as well as an internal control with a separate detection probe. Reactions were supplemented with 40 ng/mL bovine serum albumin and 400 mmol/L each dNTP. The primers and probes used were designed and published by Panning et al.^{11 (link)} (Table 1). All probes were synthesized by Tib-Molbiol (Berlin, Germany). Amplification in a 96-well ABI 7300 Real Time PCR System (Life Technology Holdings, Singapore) involved the following steps: 50 °C for 30 minutes; 95 °C for 15 minutes; and 45 cycles of denaturation at 95 °C for 15 seconds, annealing at 52 °C for 25 seconds and extension at 72 °C for 20 seconds. Fluorescence was measured at the end of each 52 °C annealing step.

The semiquantitative RT-PCR technique was used to test the reliability of the RNA-seq data. In this experiment, DEGs were randomly selected and primers were designed based on the RNA-seq contig data; information on the design of the semiquantitative RT-PCR primers is set out in Supplementary Table S1. Total RNA of R. tomentosa collected from different locations in Surat Thani and Songkhla provinces in southern Thailand was extracted using a Plant Total RNA Mini Kit (Geneaid, New Taipei City, Taiwan) according to the manufacturer’s protocol. The RT-PCR reactions contained 500 ng of RNA as template and were performed using the OneStep RT-PCR reaction kit in accordance with the manufacturer’s instructions (Qiagen, Hilden, Germany). The reaction was started at 50 °C for 30 min, followed by an initial PCR activation step at 95 °C for 15 min, then 40 cycles at 94 °C for 25 s, 60 °C for 25 s, and 72 °C for 25 s, and terminated by a 10 min incubation step at 72 °C. RT-PCR was performed to amplify a fragment of four DEG contigs and the 18S rRNA gene was amplified as a reference control. The RT-PCR products were separated using gel electrophoresis, visualized using ethidium bromide staining, and photographed to analyze expression levels using Quantity One software (Bio-Rad, Hercules, CA, USA).