A real-time PCR assay for the relative quantification of the tetrahydrocannabinolic acid (THCA) synthase gene in herbal Cannabis samples Fidelia Cascini a, *, Stella Passerotti b , Simona Martello a a Institute of Legal Medicine, University ‘Cattolica Sacro Cuore’, 00168 Rome, Italy b Laboratory ‘BioAnalisi Trentina S.r.l.’, 38068 Rovereto, TN, Italy 1. Introduction Cannabis sativa L. contains more than 420 chemical substances including at least 61 cannabinoids; the narcotic compounds [1] within (-9-tetrahydrocannabinol (THC) are responsible for the main psychoactive effects. Since the 1980s, the use of faster and more controllable methods of plant growth under optimal growing conditions in combination with the breeding of new high- performance varieties has resulted in increased yields of flower buds and increased levels of THC. Given that THC is thought to be directly derived from cannabigerolic acid (CBGA) via tetrahydro- cannabinolic acid (THCA) in all Cannabis strains [2–5], and that the conversion into THCA is catalysed by the THCA synthase enzyme, in this study, we wanted to investigate whether or not the THCA synthase gene, which codes for the enzyme [6–9], influences the production and storage of THC in a dose-dependent manner. This research hypothesis aimed to explore the genetic differences between high- and low-THC cannabis strains, starting from the idea of finding an alternative method to chemical analysis to examine forensic samples of Cannabis in order to determine the THC content. Assuming a correlation between the gene copy number and the production of THC, gene quantification could therefore be useful in forensics to distinguish the psychoactive power of seized Cannabis samples. In this study, a real-time PCR assay for the relative quantification of the gene copy number of the THCA synthase gene was validated on Cannabis samples received by our forensic laboratory. This is the first of a two-part research study concerning the THCA synthase gene in Cannabis; the second part, concerning the study of THCA synthase gene expression by the reverse transcrip- tase polymerase chain reaction (RT-PCR) in real time, did not show, for similar classes of samples, a constant correlation between the gene copy number and the gene expression data. 2. Materials and methods Genetic investigations using the real-time PCR technique were performed after the chemical analysis of 18 Cannabis samples (Table 1). Each sample was from a single plant, nine of which were seized as marijuana from the illegal drug market and the others, which were dried, were obtained from the experimental cultivation of declared potency Cannabis variety seeds. The Forensic Science International 217 (2012) 134–138 A R T I C L E I N F O Article history: Received 24 November 2010 Received in revised form 30 August 2011 Accepted 20 October 2011 Available online 16 November 2011 Keywords: Forensic science Cannabis Tetrahydrocannabinolic acid Tetrahydrocannabinol THCA synthase gene Real-time PCR Potency A B S T R A C T In this study, we wanted to investigate whether or not the tetrahydrocannabinolic acid (THCA) synthase gene, which codes for the enzyme involved in the biosynthesis of THCA, influences the production and storage of tetrahydrocannabinol (THC) in a dose-dependent manner. THCA is actually decarboxylated to produce THC, the main psychoactive component in the Cannabis plant. Assuming as the research hypothesis a correlation between the gene copy number and the production of THC, gene quantification could be useful in forensics in order to complement or replace chemical analysis for the identification and classification of seized Cannabis samples, thus distinguishing the drug- type from the fibre-type varieties. A real-time PCR assay for the relative quantification of the THCA synthase gene was then validated on Cannabis samples; some were seized from the illegal drug market and others were derived from experimental cultivation. In order to determine the gene copy number to compare high vs. low potency plants, we chose the DDCt method for TaqMan reactions. The assay enabled single plants with zero, one, and two copies of the gene to be distinguished. As a result of this first part of the research on the THCA synthase gene (the second part will cover a study of gene expression), we found no correlation between THCA synthase gene copy number and the content of THC in the herbal Cannabis samples tested. ß 2011 Elsevier Ireland Ltd. All rights reserved. * Corresponding author at: Institute of Legal Medicine, Catholic University ‘‘S. Cuore’’, L.go Francesco Vito n.1, 00168 Rome, Italy. Tel.: +39 06 30154249; fax: +39 06 35507033; mobile: +39 335 7011646. E-mail address: f.cascini@rm.unicatt.it (F. Cascini). Contents lists available at SciVerse ScienceDirect Forensic Science International jou r nal h o mep age: w ww.els evier .co m/lo c ate/fo r sc iin t 0379-0738/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2011.10.041