Selection of Reliable Reference Genes for Gene Expression Studies in the Biofuel Plant Jatropha curcas Using Real-Time Quantitative PCR

Abstract

Jatropha curcas is a promising renewable feedstock for biodiesel and bio-jet fuel production. To study gene expression in Jatropha in different tissues throughout development and under stress conditions, we examined a total of 11 typical candidate reference genes using real-time quantitative polymerase chain reaction (RT-qPCR) analysis, which is widely used for validating transcript levels in gene expression studies. The expression stability of these candidate reference genes was assessed across a total of 20 samples, including various tissues at vegetative and reproductive stages and under desiccation and cold stress treatments. The results obtained using software qBasePLUS showed that the top-ranked reference genes differed across the sample subsets. The combination of actin, GAPDH, and EF1α would be appropriate as a reference panel for normalizing gene expression data across samples at different developmental stages; the combination of actin, GAPDH, and TUB5 should be used as a reference panel for normalizing gene expression data across samples under various abiotic stress treatments. With regard to different developmental stages, we recommend the use of actin and TUB8 for normalization at the vegetative stage and GAPDH and EF1α for normalization at the reproductive stage. For abiotic stress treatments, we recommend the use of TUB5 and TUB8 for normalization under desiccation stress and GAPDH and actin for normalization under cold stress. These results are valuable for future research on gene expression during development or under abiotic stress in Jatropha. To our knowledge, this is the first report on the stability of reference genes in Jatropha.

Identification of Suitable Reference Genes for Gene Expression Normalization in Jatropha curcas L During Development and Under Stress Conditions Using Real Time Quantitative PCR

“…In addition, seedlings were subjected tofour different stress conditions, which included total stress,SA, PEG and NaCl. Zhang et al (2013) published a study aiming to select the best reference genes in J.curcas, using different parts of this plant under two different stress conditions: desiccation and cold treatments. Zhang et al (2013) recommended the use of reference genes GAPDH and EF1α for normalisation in qPCR assay in the reproductive stage.…”

“…Rocha et al (2013) were the first to identify and evaluate potential reference genes for expression studies in J. curcas for qPCR analyses, but the authors restricted their experiments to the use of developing seeds, used only six reference genes, did not include stress condition analysis, and applied only GeNorm software to normalize the data. Recently, Zhang et al (2013) published a gene expression study using reference genes in Jatropha in different tissues throughout development and only 2 under stress conditions (desiccation and cold stress treatments). The authors examined a total of 11 typical candidate reference genes using qPCR analysis, which were used for validating transcript levels in gene expression studies.…”

“…The authors used 2 target genes: JcRD29b and Jc DREB1A The expression stability of these candidate reference genes was assessed across a total of 20 samples. However, Zhang et al (2013) restricted their study to only 2 stress conditions (desiccation and cold treatments). Thus, our study consists on a better study on normalization using reference genes present in Jatropha curcas, making the present work more meticulous and complete in comparison to the two studies previous studies (Rocha et al 2013 andZhang et al 2013).…”

“…However, Zhang et al (2013) restricted their study to only 2 stress conditions (desiccation and cold treatments). Thus, our study consists on a better study on normalization using reference genes present in Jatropha curcas, making the present work more meticulous and complete in comparison to the two studies previous studies (Rocha et al 2013 andZhang et al 2013). Since qPCR results are highly dependent on the choice of appropriate reference gene(s) to control for experimental error, ideally these have to display only minor differences in their expression profile under various developmental conditions or tissue types.…”

Downregulation of LSD1 suppresses the proliferation, tumorigenicity and invasion of papillary thyroid carcinoma K1 cells

“…LSD1 has been found to specifically catalyze the demethylation of histone 3 lysine 4 (H3K4) or histone 3 lysine 9 (H3K9) (20), and to demethylate the mono-and di-methyl groups of proteins (19). It has been shown that LSD1 suppresses gene expression by targeting histone H3K4 (21,22), while gene expression is activated by targeting histone H3K9.…”