Cfx96tm real time pcr cycler

A multiplex protocol of Taq Man real-time PCR for the detection and differentiation between the MTBC members was performed to test the DNA samples extracted from EPTB human specimens, as well as positive and negative controls, following a protocol previously described by Halse et al [6 (link)]. We have used the same primers and probes of the original protocol for RD1, RD9, RD12 and ext-RD9 targets without the use of RD4 one since they are sufficient to distinguish between MTBC members as demonstrated in Table 1 [6 (link)]. Briefly, the qPCR was performed in a 25 μl final volume with Ex Taq Premix Tli RNaseH Plus (Takara, Japan) as described previously [6 (link)]. QPCR was performed on a CFX96TM real-time PCR cycler (Biorad, USA). Pure DNA was amplified in duplicate without and with a 1:5 dilution. Thorough preventive measures were taken to avoid DNA contamination during extraction and qPCR manipulation as mentioned previously [15 (link)].

The viral RNA copy number in plasma samples was determined by following a previous study^{13 (link)}. Briefly, viral RNA was extracted from 70 μl plasma using a QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol, then subjected to quantitative real-time RT-PCR using a One-Step SYBR PrimeScript RT-PCR kit II (Takara Bio, Japan). The PCR mixture was mixed with 2 μl of extracted RNA and DENV-specific primer^{46 (link)} before running on a CFX96TM real-time PCR cycler (BioRad, Hercules, CA, USA) under cycle conditions of 42 °C for 5 min, 95 °C for 10 s followed by 45 cycles of 95 °C for 5 s, 55 °C for 30 s and 72 °C for 30 s. The viral load quantity was determined by linear regression of the cycle threshold value against the known viral titers quantified by focus forming unit assay.

Total RNA was isolated using the Universal RNA Purification Kit (Roboklon, Germany). We used the optional on-column DNase (Qiagen, Hilden, Germany) digestion was conducted as defined in the protocol of the producer. Quantity and quality of the obtained RNA were checked spectrophotometrically (NanoDrop, Radnor, USA), and, in parallel, by electrophoresis on a 0.8% agarose gel. For reverse transcription of the mRNA into cDNA using the M-MuLV cDNA Synthesis Kit (New England Biolabs; Frankfurt am Main, Germany) followed the instructions of the manufacturer. The amount of RNA template was adjusted to 1 μg.
After quantitative RT-PCR (qPCR) using a CFX96^TM real-time PCR cycler (Bio-RAD, USA) expression levels of target genes were calculated with the 2^-ΔΔCt method^{71 (link)} and normalised to elongation factor 1 (EF1-α) as a house-keeping gene. Genes involved in grapevine basal immunity such as the phenylpropane phytoalexin synthesis genes phenylalanine ammonia lyase (PAL), stilbene synthase (StSy), and resveratrol synthase (RS), the transcription factor MYB14 as regulator of stilbene synthesis, and the jasmonate ZIM/tify-domain protein 1 (JAZ1) as a readout for jasmonate signalling were used. Accession numbers of these genes and the primer details are given in Table S1. Each experiment represents three biological replicates, each done as technical triplicate.