Xeno rna control

Viral RNA was extracted from 200 μl of sample (serum or lung homogenate) using a commercial kit (NucleoMag® Vet kit, Macherey-Nagel, Düren, Germany), according to the manufacturer's instructions. An exogenous internal control RNA (Xeno^TM RNA control, Applied Biosystem) was added to specimens prior to RNA extraction to verify the success of the procedure and the absence of inhibitors. The extraction was carried out on the Biosprint 96 instrument (Qiagen, Hilden, Germany), using the NucleoMag Vet 200 protocol. Nucleic acids were eluted into 100 μl of elution buffer and immediately subjected to RT-PCR or stored at −80°C until used. RNA was detected using the LSI VetMAX^(TM) PRRSV EU/NA Real-Time PCR kit (Applied Biosystem), as specified by the manufacturer.
Ten-fold serial dilutions in nuclease-free water of an EU PRRSV RNA (3.5 × 10⁵ to 35 copies/μl) were used to generate a standard curve and to quantify PRRSV RNA in the samples. Triplicates of each dilution were run in each assay. The following equation: $\text{x}=10\frac{\text{Cq}-\left(\text{yintercept}\right)}{\text{slope}},$
where x represents the genome copies per microliter and was used to transform the Cq values of the samples into estimates of genome copies of PRRSV RNA per milliliter.
The detection limit of PRRSV-1 target was nine copies of nucleic acids per reaction, corresponding to 0.642 genome copies/μl.

A total of 200 μL of sample (serum or lung suspension) was subjected to RNA extraction using a commercial kit (NucleoMag® Vet kit, Macherey-Nagel, Düren, Germany), following the manufacturer’s instructions. To control the presence of inhibitors and success of the extraction, an exogenous internal control RNA (Xeno^TM RNA control, Applied Biosystem), was included with the specimens. The extraction was carried out on the Biosprint 96 instrument (Qiagen, Hilden, Germany), using the NucleoMag Vet 200 protocol. Nucleic acids elution was performed in 100 μL of elution buffer and the LSI VetMAX ^(TM) PRRSV EU/NA Real-Time PCR kit (Applied Biosystem), was used to detect the viral RNA.
Ten-fold serial dilutions in nuclease-free water of a EU PRRSV RNA (3.5 × 10⁵ to 35 copies/μL) were used to generate a standard curve and to quantify PRRSV RNA in the samples. Triplicates of each dilution were run in each assay. The following equation: $\mathit{x}=\mathbf{10}\frac{\mathit{c}\mathit{q}-(\mathit{y}\mathit{i}\mathit{n}\mathit{t}\mathit{e}\mathit{r}\mathit{c}\mathit{e}\mathit{p}\mathit{t})}{\mathit{s}\mathit{l}\mathit{o}\mathit{p}\mathit{e}},$
where x represents the genome copies/μL, was used to transform the samples’ Cq values into estimates of genome copies of PRRSV RNA per mL of sample.
The detection limit of PRRSV-1 target was 9 copies of nucleic acids per reaction, corresponding to 0.642 genome copies/μL.