Qiaamp viral rna mini kit

The released genomic RNA from the ZIKV particles was detected by RNase digestion assay and RT–qPCR as previously described28 (link). Briefly, about 1 × 10³ plaque-forming units (p.f.u.) of ZIKV was incubated with Z2 or Z2-scr at room temperature for 2 h. The released genomic RNA from the treated ZIKV particles was then digested with micrococcal nuclease (New England BioLabs, MA) at 37 °C for 1 h. After inactivation of the residual RNase, the undigested genomic RNA in the intact viral particles was extracted using the Qiagen QIAamp Viral RNA Mini Kit (Valencia, CA) and reversed by using RT Reagent Kit (Takara Bio, Shiga, Japan). ZIKV RNA genome was quantified by SYBR PremixExTaqII (TliRNase H Plus from Takara Bio) and the Master Cycler Ep Realplex PCR System (Eppendorf, Hamburg, Germany) according to the manufacturers’ instructions. The following primers were used to detect the RNA sequences in viral genome coding precursor membrane protein (PrM), E and capsid (Cap) proteins, respectively (Fig. 3c,d): PrM F1 (5′-CTTGGACAGAAACGATGCTGGG-3′)/PrM R1 (5′-TGATGGCAGGTTCCGTACACAA-3′); E F1 (5′-TGGAGGCTGAGATGGATGG-3′)/E R1 (5′-GAACGCTGCGGTACACAAGGA-3′); and Cap F1 (5′-TCACGGCAATCAAGCCATCACT-3′)/Cap R1 (5′-GCCTCGTCTCTTCTTCTCCTT-3′).

Viral RNA was isolated from the allantoic fluid of NDV-infected chicken embryos and infected DF-1 cells using a QIAamp Viral RNA Mini kit, according to the manufacturer's instructions (TaKaRa).
To establish the rapid full-length genome cloning system of NDV, the NDV genome was divided into two fragments F1 (1–7073bp) and F2 (7054–15186bp), as illustrated in Figure 1. The F1 and F2 fragments of each NDV were transcribed from viral RNA with two pairs of specific primers and then amplified by using a high-fidelity RT-PCR Reagent kit (TaKaRa). In the first step, the F1 fragment was generated by RT-PCR with specific primers F1 F and F1 R and cloned into the corresponding modified vector, which was linearized by PCR with specific primers F1 Vet up and F1 Vet down from the plasmid pZM10-RFP (Genbank accession no. JX390609) by using PrimeSTAR® GXL DNA Polymerase (TaKaRa), resulting in the production of subclones pVG/GA-F1, pLaSota-F1, and pF48E9-F1, respectively. Subsequently, the full-length genome plasmids pVG/GA, pLaSota, and pF48E9 were obtained by cloning the F2 fragment amplified with the primers F2 F and F2 R into the pVG/GA-F1, pLaSota -F1, and pF48E9-F1 vectors linearized by PCR with specific primers F1 Vet up and F1 R, respectively.

HIV-1 RNA was extracted using the QIAamp viral RNA mini kit and reverse transcribed to cDNA with the RT-PCR Prime Script Kit from Takara. Then, 2 μL cDNA was used in a 20 μL qRT-PCR reaction using the Takara PCR Master Mix with the specific HIV-1 P17 gene primers (5′-TCTCGA CGCAGG ACTCG-3′ and 5′-TACTGA CGCTCT CGCACC-3′) and a TaqMan probe (5′-FAM-CTCTCT CCTTCT AGCCTC-MGB-3′), in the following conditions: 1 cycle of 50 °C for 2 min, 1 cycle of 95 °C for 10 min and 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The HIV-1 viral titer was determined via comparison with a standard curve generated using RNA extracted from a serially diluted reference HIV-1 viral stock.