Computational protocol: Ethylene Response Factor 6 Is a Regulator of Reactive Oxygen Species Signaling in Arabidopsis

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Protocol publication

[…] For RNA extraction plant samples were collected after the treatments, at the indicated time points, and were immediately immersed in liquid nitrogen and stored at -80°C. After grinding of twenty 4 weeks-old plants in liquid nitrogen to a fine powder, a representative sample of approximately 70 mg plant tissue was used for RNA extraction using the SV Total RNA Isolation System (Promega). RNA integrity was tested by gel electrophoresis and quantity measured by NanoDrop spectrophotometer (ND-1000 spectrophotometer). The same amount of RNA (from 1000 to 2000 ng) was used for cDNA synthesis in each experiment. SuperScript III reverse transcriptase (Invitrogen) was used for cDNA synthesis according to the supplier’s instructions. The Primer Express 2.0 software (Applied Biosystems) and DNA sequences, as templates, from the TAIR website (http://www.arabidopsis.org/) were used for primer design (). The primers were designed to amplify 100–150 bp close to the 3′ end of the gene. The specificity of the forward and reverse primers to the candidate gene was checked using the NCBI-BLAST website (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) and melting curve analysis following qRT-PCR. Primer efficiencies were incorporated into the data analysis and β-actin genes of Arabidopsis, β-actin-2 (At3g18780), β-actin-7 (At5g09810), and β-actin-8 (At1g49240) primers were used as an internal control for normalization. Briefly, qRT-PCR was performed in optical 384-well plates using an ABI7900 HT Sequence Detection System (Applied Biosystems, Warrington, UK). Each reaction contained 6 µl of 2× SYBR Green Master Mix reagent (Applied Biosystems), 10 ng cDNA and forward and reverse gene specific primers at a concentration of 250 nM. The thermal profile comprised 95°C for 10 min followed by 45 cycles of 95°C for 15 s and 60°C for 1 min. Data were analyzed using SDS2.2 software (Applied Biosystems) and Microsoft Excel. Amplification plots were analyzed to provide cycle threshold values (Ct) using an Rn threshold of 0.3 for each primer pair-cDNA combination. PCR primer efficiency (E value) of each primer pair was calculated by linear regression analysis for each reaction. Absolute gene expression levels relative to actin reference genes was calculated for each cDNA sample using the equation: relative ratio gene/actin = (Egene–(Ct gene))/(Eactin–(Ct actin)). Student’s t-test or two-way ANOVA (GraphPad Prism 5) was used to determine statistical significance. […]

Pipeline specifications

Software tools Primer Express, BLASTN
Databases TAIR
Application qPCR
Organisms Arabidopsis thaliana
Chemicals Anthocyanins, Calcium, NADP, Oxygen