Computational protocol: Analysis of the transgenerational iron deficiency stress memory in Arabidopsis thaliana plants

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Protocol publication

[…] RNA was extracted from seedlings (after 14 days from germination) with TRIzol® reagent (Life Technologies, Carlsbad, CA, USA), by following manufacturer’s instructions. qRT-PCR was carried out using the iTaqTM universal SYBR® Green one-step kit (Bio-Rad Laboratories, Hercules, CA, USA) using a Rotor-Gene 6000 PCR apparatus (Corbett Robotics Pty Ltd., Brisbane, QLD, Australia). Reaction conditions were as follows: reverse transcription at 50°C for 10 min, denaturation at 95°C for 1 min, and 40 cycles of 95°C 15 s and 60°C 30 s. After the amplification a melting reaction was carried out. The expression profiles of A. thaliana TFIIS gene (TAIR annotation At2g38560) and TFIIS-like gene (TAIR annotation At5g09850; ) were obtained by using β-Tubulin (TAIR annotation At1g20010) and Ubiquitin4 (TAIR annotation At5g20620) as reference genes. Primers were designed with the Primer Design online tool Primer3 and checked with an OligoAnalyzer tool (Integrated DNA Technologies). For each oligonucleotide set, a no-template water control was used. qRT-PCR data analysis was carried out according to . Statistical analysis was performed with SigmaStat (Systat Software, Richmond, CA, USA). […]

Pipeline specifications

Software tools Primer3, OligoAnalyzer
Databases TAIR
Application qPCR
Organisms Arabidopsis thaliana
Diseases Deficiency Diseases
Chemicals Iron, Oxygen