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

Sven K Delaney,Gregor W Schmidt,Teresa Docimo,John C D'Auria,Katrin Luck

doi:10.12688/f1000research.2-37.v1

Abstract

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. Erythroxylum coca produces and accumulates high amounts of the 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, TPB1, Pex4, Pp2aa3). The expression of these genes was measured in seven 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 Ec10131 and TPB1 are the most appropriate internal reference genes in leaves (where the majority of cocaine is produced), while Ec10131 and Ec6409 are the most suitable internal reference genes across all of the tissues tested.

Highlights

Erythroxylum coca has been cultivated by humans for more than 8000 years and has been selected for high-level production of cocaine, a pharmacologically active tropane alkaloid
We evaluate the stability of nine candidate reference genes (Ec6409, Ec10131, Ec11142, Actin, Adenine phosphoribosyl transferase 2 (APT2), EF1α, TPB1, Pex[4] and Pp2aa3) in a variety of E. coca tissues
The organs used for RNA extraction and Quantitative real-time reverse-transcription PCR (qRT-PCR) analysis were obtained from four-month old E. coca plants grown from rooted cuttings

Summary

Introduction

Erythroxylum coca has been cultivated by humans for more than 8000 years and has been selected for high-level production of cocaine, a pharmacologically active tropane alkaloid. Despite the socioeconomic importance of cocaine and other tropane alkaloids, the molecular basis for the biosynthesis of the tropane nucleus remains unknown. E. coca is emerging as a model for the investigation of tropane alkaloid synthesis[2,3,4], and shows high-level, localized tropane alkaloid production and storage in its leaf tissue[3,4]. We have performed metabolic and enzymatic studies to identify the molecular and biochemical basis of tropane alkaloid biosynthesis in E. coca, and have developed a number of genomic tools such as expressed sequence tag (EST) libraries and 454 sequence databases[2,3,4]. Quantitative real-time reverse-transcription PCR (qRT-PCR) would be a further source of information on candidate tropane alkaloid biosynthesis genes in the different tissues of the coca plant

Methods

Results

Conclusion