Real-time quantitative PCR (RT-qPCR) has greatly improved the quality of measurements of expression levels of target genes in biological samples, as a result of its high sensitivity, specificity and cost-efficiency. It has been widely used for gene expression analysis. However, the accuracy of RT-qPCR analysis can be significantly affected by several factors, including RNA quality, the quantity of cDNA and the selection of reference genes. To achieve high accuracy, a reference gene should have a relatively stable expression level in distinct biological samples. Sacha inchi (Plukenetia volubilis) is a promising oilseed crop known for its polyunsaturated fatty acid (PUFA)-rich seeds. However, suitable reference genes for RT-qPCR have not been identified in Sacha inchi.
Prof. XU Zengfu and his team of Xishuangbanna Tropical Botanical Garden (XTBG) selected a total of twelve candidate reference genes (18S, ACT, CYC, EF1α, GAPDH, PLA, RPII, RPS13, TEF2, TUB, UBL and UCE) to normalize the gene expression levels in Sacha inchi using RT-qPCR. The expression stabilities of the twelve candidate reference genes were estimated in various tissues and developmental stages of Sacha inchi. Four statistical algorithms (delta cycle threshold (ΔCt), BestKeeper, geNorm, and NormFinder) were used to evaluate the stability of the expression levels of these twelve candidate reference genes.
The ubiquitin-conjugating enzyme (UCE), actin (ACT) and phospholipase A22 (PLA) genes were found to be the most stable genes in seedlings. For roots, stems, leaves, flowers, and seeds from adult plants, 30S ribosomal protein S13 (RPS13), cyclophilin (CYC) and elongation factor-1alpha (EF1α) were recommended as reference genes for RT-qPCR. During the development of reproductive organs, PLA, ACT, and UCE were the optimal reference genes for flower development, whereas UCE, RPS13, and RNA polymerase II subunit (RPII) were optimal for seed development. For analyses of the entire growth cycle of Sacha inchi, the three best reference genes are CYC, RPS13 and UCE.
The study entitled “Selection of Reliable Reference Genes for Gene Expression Studies of a Promising Oilseed Crop, Plukenetia volubilis, by Real-Time Quantitative PCR” has been published in International Journal of Molecular Sciences.
Sacha inch (Plukenetia volubilis) (Image by XTBG)
