Computational protocol: ZINC-INDUCED FACILITATOR-LIKE family in plants: lineage-specific expansion in monocotyledons and conserved genomic and expression features among rice (Oryza sativa) paralogs

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Protocol publication

[…] We used only specific Affymetrix probes for rice ZIF genes (Additional File ) to analyze expression data from GENEVESTIGATOR (http://www.genevestigator.com) []. Only high quality arrays were used. [...] For quantitative RT-PCR analysis (qPCR), the synthesized first strand cDNA from each time point was diluted 100 times. qPCR was carried out in an Applied Biosystems StepOne real-time cycler. All primers (listed in Additional File ) were designed to amplify 100-150 bp of the 3'-UTR of the genes and to have similar Tm values (60 ± 2°C). Reaction settings were composed of an initial denaturation step of 5 min at 94°C, followed by 40 cycles of 10 s at 94°C, 15 s at 60°C, 15 s at 72°C; samples were held for 2 min at 60°C for annealing of the amplified products and then heated from 60 to 99°C with a ramp of 0.3°C/s to provide the denaturing curve of the amplified products. qPCRs were carried out in 20 μl final volume composed of 10 μl of each reverse transcription sample diluted 100 times, 2 μl of 10X PCR buffer, 1.2 μl of 50 mM MgCl2, 0.1 μl of 5 mM dNTPs, 0.4 μl of 10 μM primer pairs, 4.25 μl of water, 2.0 μl of SYBR green (1:10,000, Molecular Probe), and 0.05 μl of Platinum Taq DNA polymerase (5 U/μl, Invitrogen®, Carlsbad, CA, USA). Obtained data were analyzed using the comparative Ct (threshold cycle) method []. The PCR efficiency from the exponential phase (E) was calculated for each individual amplification plot using the LinRegPCR software []. In each plate, the average of PCR efficiency for each amplicon was determined and used in further calculations. Ct values for all genes were normalized to the Ct value of UBQ5 []. The equation Q0 target gene/Q0 UBQ5 = [(Eff UBQ5) Ct UBQ5/(Eff target gene) Ct target gene], where Q0 corresponds to the initial amount of transcripts, was used for normalization []. Each data point corresponds to three true biological replicate samples. […]

Pipeline specifications

Software tools Genevestigator, LinRegPCR
Applications qPCR, Transcription analysis
Organisms Oryza sativa, Homo sapiens
Diseases Deficiency Diseases
Chemicals Iron, Zinc