1 step rt pcr kit

Total RNA was isolated from siRNA-treated cells using either the miRNeasy kit (Qiagen; qRT-PCR) or NucleoSpin RNA kit (Macherey and Nagel; qualitative RT-PCR) including a DNase digest. Equal amounts of RNA from control or USPL1-knockdown cells were subjected to qualitative RT-PCR using the 1-Step RT-PCR kit from Qiagen with gene-specific primers (see supplementary material Table S5) and analysed by ethidium bromide agarose gel electrophoresis. qRT-PCR was performed on 0.4 ng of RNA per reaction (or 4 ng RNA in the case of U2pre-snRNA) in duplicate using the QuantiFast RT-PCR SYBR-Green Mix (Qiagen) in a Roche Light Cycler 480. Primers (see supplementary material Table S6) were tested for their PCR efficiency and the formation of primer dimers under qRT-PCR conditions.

The extracted nucleic acids (from a Roche MagNApure compact automated system) were used for 1-step real-time PCR for RSV detection, using a Qiagen 1-step RT-PCR kit. Real-time RT-PCR assays were performed with primers and probes for RSV, using the CDC protocol [11 ]. All assays were run with positive controls (RSV-RNA), negative controls (nuclease-free water), and internal positive controls (RNAseP) for quality-control purposes.