Computational protocol: Barley disease susceptibility factor RACB acts in epidermal cell polarity and positioning of the nucleus

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Protocol publication

[…] Gene expression analysis was carried out by reverse transcription quantitative real-time PCR (RT–qPCR) in a Mx3005P cycler (Agilent Technologies, Santa Clara, CA, USA) using the Maxima SYBR Green qPCR master mix (2×) (Thermo Fisher Scientific, St. Leon-Rot, Germany). Reactions were performed in duplicate with 10ng of cDNA and 330nM forward and reverse primer each in a final volume of 10 µl. Expression values of defence genes and RACB were normalized to a barley housekeeping ubiquitin (HvUBI) () using primer efficiency correction as suggested by . The program consisted of an initial step at 95 °C for 10min and 95 °C for 30s, followed by 40 cycles at 55 °C for 30s and at 72 °C for 1min. The melting curve analysis was performed at 55–95 °C. All primers () were designed using Primer3 software () and were checked for specificity using the Basic Local Alignment Search Tool (BLAST) and therein with nucleotide blast against the H. vulgare database (http://blast.ncbi.nlm.nih.gov), and amplicon size assessment in agarose gels before running RT–qPCR. […]

Pipeline specifications

Software tools Primer3, BLASTN
Application qPCR
Organisms Hordeum vulgare, Flavobacterium sp.