Plant Science 183 (2012) 9–13 Contents lists available at SciVerse ScienceDirect Plant Science journal homepage: www.elsevier.com/locate/plantsci Trichome isolation with and without fixation using laser microdissection and pressure catapulting followed by RNA amplification: Expression of genes of terpene metabolism in apical and sub-apical trichome cells of Artemisia annua L. Linda Olofsson, Anneli Lundgren, Peter E. Brodelius ∗ School of Natural Sciences, Linnaeus University, SE-39182 Kalmar, Sweden article info Article history: Received 12 September 2011 Received in revised form 21 October 2011 Accepted 29 October 2011 Available online 4 November 2011 Keywords: LMPC Artemisia annua Trichomes qPCR Gene expression Artemisinin biosynthesis abstract The aim of this project was to evaluate the effect of fixation on plant material prior to Laser Microdis- section and Pressure Catapulting (LMPC) and to identify an appropriate method for preserving good RNA quality after cell isolation. Therefore, flower buds from Artemisia annua L. were exposed to either the fixative formaldehyde or a non-fixative buffer prior to cell isolation by LMPC. Proteinase K was used after cell isolation from fixed plant tissue, in an attempt to improve the RNA yield. The ability to detect gene expression using real-time quantitative PCR with or without previous amplification of RNA from cells iso- lated by LMPC was also evaluated. Conclusively, we describe a new technique, without fixation, enabling complete isolation of intact glandular secretory trichomes and specific single trichome cells of A. annua. This method is based on LMPC and preserves good RNA quality for subsequent RNA expression studies of both whole trichomes, apical and sub-apical cells from trichomes of A. annua. Using this method, expres- sion of genes of terpene metabolism was studied by real-time quantitative PCR. Expression of genes involved in artemisinin biosynthesis was observed in both apical and sub-apical cells. © 2011 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Laser Microdissection and Pressure Catapulting (LMPC) is a tech- nique that allows selected cells to be harvested from complex tissue sections while being viewed on a computer screen connected to a microscope [1]. LMPC allows isolation of specific tissues, single cells or even organelles thereby excluding adjacent cells that would oth- erwise contaminate the sample [2]. Additionally, LMPC avoids any mechanical contact with the sample, thereby reducing the risk of contamination [1–3]. Further, negligible heat is transferred to the tissue or cell while catapulted by the laser since the laser is only directed at the sample for about 1 ns and consequently, the recovery of nucleic acids or proteins is not impaired by elevated tempera- Abbreviations: ADS, amorpha-4,11-diene synthase; ALDH1, aldehyde dehy- drogenase 1; CPR, cytochrome P450 reductase; CPS, caryophyllene synthase; CYP71AV1, amorphadiene-12-hydroxylase; DBR2, artemisinic aldehyde 11(13) reductase; DXR, 1-deoxy-d-xylulose 5-phosphate reductase; DXS, 1-deoxy-d- xylulose 5-phosphate synthase; ECS, epi-cedrol synthase; FDS, farnesyl diphosphate synthase; GAS, germacrene A synthase; GST, glandular secretory trichomes; GUS, -glucuronidase; HDR, hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase; HMGR, 3-hydroxy-3-methyl-glutaryl-CoA reductase; IDI, isopentenyl diphosphate isomerase; LMPC, Laser Microdissection and Pressure Catapulting; LS, linalool syn- thase; RED1, dihydroartemisinic aldehyde reductase; SQS, squalene synthase. ∗ Corresponding author. Tel.: +46 480 447358; fax: +46 480 446262. E-mail address: peter.brodelius@lnu.se (P.E. Brodelius). ture and living cells can be recultivated following LMPC. The LMPC technique is used to study cell type-specific expression at the DNA, RNA and protein levels [4]. Glandular secretory trichomes (GSTs) of Artemisia annua L. have received increasing attention since the biosynthesis of artemisinin has been proposed to be located to such trichomes. Artemisinin is currently being used worldwide in the treatment of malaria. Though, given that the plant produces relatively small amounts of artemisinin, there is a worldwide shortage of the drug and there is an ongoing pursuit in learning more about the artemisinin produc- ing trichomes and thereby increasing the possibility to enhance the production of the antimalaria drug in the plant. For that purpose GSTs from A. annua were isolated by LMPC and gene expression was studied by real-time quantitative PCR (qPCR) to identify enzymes that are expressed in such trichomes. Previously, trichome isola- tion from A. annua has been reported using cell disrupters, where the trichomes are isolated mechanically by glass beads and there- after separated from other cells and tissue fragments by sequential filtration through nylon mesh filters [5–8]. The disadvantage with cell disrupter methods is that the final trichome preparation has approximately 10% impurities [5]. With our trichome isolation method we isolate only selected cells, without contamination from adjacent cells. This together with LMPC being a non-contact method greatly reduces contamination risks. The quality of the specimen is of great importance for accurate validity of the data obtained from a study. The aim is to preserve 0168-9452/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2011.10.019