Hiscript 2 one step qrt pcr sybr green kit

Total RNAs were isolated from fresh LUAD tissues using Trizol reagent (Takara Bio Inc., China). HiScript II One Step qRT-PCR SYBR Green Kit (Vazyme, China) was used in qRT-PCR analysis. The sequences of all primers used are shown in Table 3. All primers were designed and purchased from Tsingke (Wuhan, China).

Extracellular viral RNA was extracted from supernatants of SADS-CoV-inoculated cells at different time points using the High Pure viral RNA kit (Roche, Basel, Switzerland). Intracellular viral RNA was extracted from cell lysate using a magLEAD nucleic acid purification system (Precision System Science Co., Ltd., Japan). Specific primers for the RdRp gene of SADS-CoV (forward, 5′-GCGATGAGATGGTCACTAAAGG-3′; reverse, 5′-GGAATACCCATACCTGGCATAAC-3′) were designed according to the reference sequence (GenBank accession number MF094681). For SADS-CoV strand-specific real-time RT-PCR, RdRp-forward primer and RdRp-reverse primer were used to synthesize viral minus-strand and plus-strand RNA, respectively. A real-time one-step quantitative RT-PCR assay was used to determine the SADS-CoV genomic RNA using the HiScript II one-step qRT-PCR SYBR green kit (Vazyme, China) as described previously (12 (link)).

The concentration and purity of all RNA samples obtained from in vitro and in vivo infection experiments were measured with a Qubit 4.0 Fluorometer (Thermo Fisher Scientific, Loughborough, UK). Next, 20 ng of total RNA was used to directly amplify various genes of TLRs and proinflammatory mediators with the HiScript^® II One Step qRT-PCR SYBR Green Kit (Vazyme biotech, Nanjing, China). The PCR amplification protocol for all RNA samples was 15 min at 50 °C, followed by 30 s at 95 °C and 40 cycles of denaturing for 10 s at 95 °C and 30 s at 60 °C. A melting curve step (15 s at 95 °C followed by 60 s at 60 °C and 15 s at 95 °C) was added at the end of each amplification. Each 20 μL reaction contained forward and reverse primers at a concentration of 200 nM. The primers of the TLRs and proinflammatory mediators were listed in Table 1. The gapdh gene (encoding glyceraldehyde 3-phosphate dehydrogenase) was used as a normalizing gene. Non-infected primary astrocytes, BV2 cells, and mice were used as the calibration reference in the corresponding analyses, respectively. The quantitation of the differences between groups was calculated using the 2^−ΔΔCt method. The transcription values were expressed as mean + standard deviation.

Tumor tissue was collected following treatment and extracted with TRIzol™ Plus RNA Purification kit (Invitrogen, USA) according to the manufacturer’s instructions. RNA samples were analyzed using the HiScript II One Step qRT-PCR SYBR Green kit (Vazyme, China). Table S1 gives the sequences of RT-qPCR primers.