Computational protocol: Transcriptome profile analysis of flowering molecular processes of early flowering trifoliate orange mutant and the wild-type [Poncirus trifoliata (L.) Raf.] by massively parallel signature sequencing

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Protocol publication

[…] To assign putative functions to differentially expressed genes between MT and WT, annot8r program was run locally to BLAST against a reference database that stores UniProt entries, their associated Gene Ontology (GO), Enzyme Commission (EC), and Kyoto Encyclopaedia of Genes and Genomes (KEGG) annotation []. The GO categorization results were expressed as three independent hierarchies for biological process, cellular component, and molecular function []. The biological interpretation of the differentially expressed genes was further completed by assigning them to metabolic pathways using KEGG []. For the identification of pathways significantly affected by the mutation, we focused on the metabolite pathways with at least three affiliated genes. [...] Thirty genes were chosen for confirmation by real-time quantitative RT-PCR with SYBR green I chemistry (QIAGEN, Germany). Primers for these genes were designed with the Primer Express software (PE Applied Biosystems, USA) and tested to ensure amplification of single discrete bands with no primer-dimers. Product size was about 180 bp. Total RNA (3 mg) was treated with 3 U of DNase (Promega, USA) and then used in first-strand synthesis with an oligo (dT) primer (20-mer) and reverse transcriptase according to the manufacturer's instructions. For real-time PCR, an amount of cDNA corresponding to 25 ng of input RNA was used in each reaction. Reactions were performed with the SYBR Green PCR Master Mix and analyzed in the ABI 7500 Real-Time System. Real-time PCR products were amplified with 1 μl of template of the RT reaction mixture, 10 μl of 2 × SYBR Green Master Mix, and 0.5 μl of forward and reverse primer (10 μmol/μl), with water to a final volume of 20 μl. The levels of gene expression were analyzed with ABI 7500 Sequence Detection System Software (PE Applied Biosystems) and normalized with the results of β-actin. Real-time quantitative PCR was performed in four replicates for each sample, and data were indicated as means ± SD (n = 3). […]

Pipeline specifications

Software tools annot8r, Primer Express
Databases KEGG KEGG Annotation
Applications qPCR, Transcription analysis
Diseases Machado-Joseph Disease