Computational protocol: Identification and testing of reference genes for Sesame gene expression analysis by quantitative real-time PCR

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Protocol publication

[…] Ten housekeeping sesame genes, including SiACT, SiUBQ6, SiTUB, Si18S rRNA, SiEF1α, SiCYP, SiHistone, SiDNAJ, SiAPT and SiGAPDH, were selected as candidate reference genes. Three functional genes of late embryogenesis abundant protein (SiLEA), starch synthase (SiSS) and glycosyl hydrolase family protein (SiGH) were selected for reference gene validation. Si18S rRNA sequence was obtained from NCBI data (Accession number: AJ236041). The sequences of other nine reference genes and three validation genes were obtained from our sesame RNA-seq transcriptome dataset ( accession numbers JP631635-JP668414) and compared with the AGI (Arabidopsis Genome Initiative) protein database using BLASTX (; Table ) with E-value cut-off of 1E−20 as ‘significant matches’. [...] qRT-PCR primers for the above thirteen genes were designed using Primer Express 3.0 (ABI) with the melting temperature between 60 and 62 °C and a primer length of 20–26 bp. The length of amplicons ranged from 100 to 251 bp with high polymerization efficiency, which minimized the RNA integrity impact (Fleige and Pfaffl ). Meanwhile, specific probes of ten candidate genes with 5′FAM and 3′BHQ1 fluorescence radicals were designed with the melting temperature between 68 and 71 °C, a length of 24–30 bp and about 50 % GC content (Table ). [...] To assay the gene expression variability in sesame, qRT-PCR was conducted with an Eppendorf Mastercycler ep Realplex 2.2 Detection System. The PCR reaction volume was 30 μL containing 2.0 μL of diluted cDNA, 0.2 μM of each primer, 0.1 μM probe, 1× PCR buffer, 50 μM of each dNTP and 1.0 U Platinum Taq DNA polymerase (Invitrogen). Reaction mixtures were incubated for 2 min at 37 °C, 5 min at 95 °C, followed by 40 amplification cycles of 15 s at 95 °C and 60 s at 60 °C. All samples were amplified in triplicate times. A negative control without cDNA template was also done at the same time. A standard curve for each gene was generated using tenfold serial dilutions of pooled cDNAs (data not shown). The efficiency of the thirteen pairs of primers in qRT-PCR was calculated using LinRegPCR (Ramakers et al. ). To determine their amplicon specificity, electrophoresis analysis of the PCR products was also carried out. Expression levels of the 13 genes in all samples were determined by their cycle threshold values (Cts). [...] Three publicly available software tools, i.e., geNorm (Vandesompele et al. ), Normfinder (Andersen et al. ) and Bestkeeper (Pfaffl et al. ), were used for expression stability determination of the ten genes in sesame. […]

Pipeline specifications

Software tools BLASTX, Primer Express, LinRegPCR, NormFinder, BestKeeper
Applications RNA-seq analysis, qPCR
Organisms Sesamum indicum