Selection and validation of reference genes for quantitative gene expression studies in

Real-time quantitative PCR is a powerful technique for the investigation of comparative gene expression, but its accuracy and reliability depend on the reference genes used as internal standards. Only genes that show a high level of expression stability are suitable for use as reference genes, and these must be identified on a case-by-case basis. produces and accumulates high amounts of the Erythroxylum coca pharmacologically active tropane alkaloid cocaine (especially in the leaves), and is an emerging model for the investigation of tropane alkaloid biosynthesis. The identification of stable internal reference genes for this species is important for its development as a model species, and would enable comparative analysis of candidate biosynthetic genes in the different tissues of the coca plant. In this study, we evaluated the expression stability of nine candidate reference genes in ( , , , , , , E. coca Ec6409 Ec10131 Ec11142 Actin APT2 EF1α , , ). The expression of these genes was measured in seven TPB1 Pex4 Pp2aa3 tissues (flowers, stems, roots and four developmental leaf stages) and the stability of expression was assessed using three algorithms (geNorm, NormFinder and BestKeeper). From our results we conclude that and Ec10131 are the most appropriate internal reference genes in leaves (where the TPB1 majority of cocaine is produced), while and are the most Ec10131 Ec6409 suitable internal reference genes across all of the tissues tested. 1,2* 1,3* 1 4