Quantitect sybr green pcr master

Viral or total cellular DNA was extracted with a DNAeasy kit (Qiagen, Courtaboeuf, France) and the DNA concentration and purity were determined using a Nanodrop spectrophotometer. Real-time PCR was performed using different dilutions of the purified total DNA and the QuantiTect SYBR green PCR master (Qiagen, Courtaboeuf, France) with a LightCycler 96 thermal cycler (Roche Applied Science, Meylan, France), using the E11L primers previously described [51 (link)]. For relative quantification, the 2^−ΔΔCt method was used [52 (link)]. The quantification of copies per millilitre was performed against a standard curve of WR virions. The number of viral DNA copies was evaluated either by Nanodrop (1 ng DNA viral DNA = 4.74 × 10⁶ copies) or directly by the qPCR assay.

RNA was isolated from infected and non-infected neuronal/glial cells and in cultures enriched in neurons (En-Ne) or astrocytes (En_As). Cells were lysed using the NucleoMag^® 96 RNA kit (Macherey Nagel, Hoerdt, France) and RNA was extracted with a King Fisher Duo automat (Fisher Scientific, Illkirch, France) following the manufacturer’s instructions. Two hundred and fifty ng (cultures without enrichment) or 150 ng (cultures with enrichment) of RNA were used to synthesize cDNA with the SuperScript™ II Reverse Transcriptase kit (Thermo Fisher Scientific, Illkirch, France). Real-time PCR was performed using 2 µL of cDNA and QuantiTect SYBR green PCR master (Qiagen, Courtaboeuf, France) with a LightCycler 96 instrument (Roche Applied Science, Meylan, France), for a total volume of 20 µL of reaction mixture. For relative quantification, the −2^ΔΔCt method was used [40 (link)]. The reference gene was HPRT1. Primers pairs are listed in Table S1. * indicates that primers were designed using “primer designer” from the website: https://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi, accessed on 16 August 2021. PCR efficiency determined for each pair of primers was greater than 97% with the exception of TNFSF10 (=91%).