Rotor gene q series software q 2

Transcript levels of genes were determined by quantitative PCR with the SYBR Green method on a Rotor-Gene Q (Qiagen). A 15 μl reaction mixture contained the following components: 5 μl cDNA, 1 μl of each primer (5 μM stock), 1.5 μl buffer, 0.3 μl dNTPs (10 mM each), 5.75 μl water, 0.3 μl JumpStart Taq DNA polymerase and 0.15 μl CYBR Green (1:400 dilution of purchased stock solution of ABsolute™ QPCR SYBR Green Fluorescein Mix, ABgene). The thermal cycling conditions used were 95 °C for 6 min followed by 40 cycles of 95 °C for 15 s, 58 °C for 30 s, and 72 °C for 20 s, followed by a melt cycle with 1 °C increments for 5 s each from 56 to 96 °C. The analysis of melting curves, measurement of primer pair efficiencies, and determination of cycle threshold values, including the calculation of the mean normalized expression of the genes, was conducted using the Rotor-Gene Q Series Software Q 2.0.2 (Qiagen) and the Q-Gene software. The mRNA levels of the studied genes were normalized using the comparative C_T method, using the expression of one or two reference genes (actin and ribosomal protein L19, RPL19) as internal standard (Maroufi et al., 2010 (link); van Arkel et al., 2012 (link)). Primer efficiency was considered valid when calculated efficiency was between 90 and 110% with 100% as an optimum.

For cDNA synthesis, 350 ng of grapevine total RNA was reverse transcribed using the dART cDNA synthesis kit (Roboklon) as described in Höll et al. [48 (link)]. Transcript analysis of genes of interest (GOI) during P. viticola infection were determined by qPCR with the SYBR Green method on a Rotor-Gene Q (Qiagen). The PCR reaction mix (15 μl) contained cDNA (1.2 ng), primer (10 μM each), dNTP mix (10 mM each) (Sigma Aldrich), JumpSTART polymerase (2.5 U/μl) (Sigma Aldrich), 0.15 μl from 1:40 dilution SYBR Green in H₂O (ABsolute™ QPCR SYBR® Green Fluorescein Mix; 1:10 in DMSO; ABgene) and nuclease free water. The thermal cycling conditions used were 95 °C for 6 min followed by 40 cycles of 95 °C for 15 s, 58 °C for 30 s, and 72 °C for 20 s, followed by a melt cycle with 1 °C increments (5 s) from 56 to 96 °C. The primer efficiency was tested with cDNA dilutions of samples. Normalization against the reference genes VvUbiquitin, VvEF1α and VvGAPDH [78 (link)] was conducted as described by Pfaffl et al. [79 (link)]. The Rotor-Gene Q Series Software Q 2.0.2 (Qiagen) and the Q-Gene software [80 ] were used for analyzing melt curves and measurement of primer pair efficiency. Gene-specific oligonucleotide sequences are shown in Additional file 6 and representative melting curves are presented in Additional file 7.