Computational protocol: Genome-Wide Investigation and Expression Analyses of WD40 Protein Family in the Model Plant Foxtail Millet (Setaria italica L.)

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Protocol publication

[…] To elucidate the tissue-specific expression profile of SiWD40 genes, the Setaria italica Illumina RNA-HiSeq reads from 4 tissues namely spica, stem, leaf and root were retrieved from European Nucleotide Archive [SRX128226 (spica); SRX128225 (stem); SRX128224 (leaf); SRX128223 (root)] . The RNA-seq data was then filtered by NGS toolkit to remove low quality reads and was mapped onto the gene sequences of Setaria italica by CLC Genomics Workbench v.4.7.1 (http://www.clcbio.com/genomics). The number of reads mapped was normalized by RPKM (reads per kilobase per million) method. The heat map showing tissue specific expression was generated on the RPKM value for each gene in all the tissue samples using TIGR MultiExperiment Viewer (MeV4) software package , . [...] Total RNA was isolated by following the procedure described by Longeman et al. and treated with RNase-free DNase I (50 U/µl; Fermentas, USA) for removing DNA contamination. The quality and purity of the preparations were determined at OD260:OD280 nm absorption ratio (1.8–2.0) and the integrity of the preparations was determined by resolving in 1.2% agarose gel containing formaldehyde. About 1 µg total RNA was reverse transcribed to first strand cDNA using random primers by Protoscript M-MuLV RT (New England Biolabs, USA) following manufacturer’s instructions . The qRT-PCR primers were designed using Primer Express 3.0 software (PE Applied Biosystems, USA) with default parameters (). qRT-PCR was carried out in three technical replicate for each biological duplicate by one step real time PCR system of Applied Biosytems (USA). The PCR mixtures and reactions were used as described previously by Kumar et al.21 Melting curve analysis (60 to 95°C after 40 cycles) and agarose gel electrophoresis were performed to check amplification specificity for absence of multiple amplicons or primer dimers . A constitutive Act2 gene-based primer was used as endogenous control. The amount of transcript accumulated for SiWD40 genes normalized to the internal control Act2 were analyzed using 2−ΔΔCt method cDNA synthesis. The PCR efficiency which is dependent on the assay, performance of the master mix and quality of sample, was calculated as: Efficiency = 10 (−1/slope) − 1 by the software itself (Applied Biosystems). […]

Pipeline specifications

Software tools CLC Genomics Workbench, CLC Assembly Cell, Primer Express
Databases ENA
Applications RNA-seq analysis, qPCR
Organisms Zea mays, Sorghum bicolor, Setaria italica, Oryza sativa