 a sufficient amount of material for analysis using this technique is laborious. Moreover it works better on larger, well-developed syncytial cells as compared to syncytial tissues during the early stages of development. An alternative and promising technique for collecting pure cells is laser capture microdissection (LCM).
LCM is becoming a popular tool among cell biologists because it allows isolation of RNA, DNA, proteins and metabolites from a heterogeneous cell population. Current LCM techniques are based on two principles. In the "touch-free" approach, cells identified under a microscope are excised by a laser beam and removed from their tissue context by gravity or by catapulting them away with a photonic pulse. Alternatively, cells are fused to a plastic membrane and are subsequently removed mechanically from the remaining tissue (reviewed in Ludwig and Hochholdinger [22]).
Successful LCM requires good morphological preservations of tissues. Furthermore, isolated nucleic acids (RNA or DNA), proteins or metabolites must be of high quality to ensure their utility for downstream applications. Therefore, tissue preparation is the crucial step towards successful RNA isolation of samples generated by LCM. Since LCM requires micro-sections of 2-20 µm, samples must be fixed and embedded prior to sectioning and mounting them on the slides. During this multistep procedure, RNA integrity remains at high risk of degradation due to the presence of RNases. Samples for immuno-histological analyses are generally fixed in paraformaldehyde and embedded in paraffin to ensure wellpreserved tissue morphology. In contrast, ethanol:acetic acid (EAA) solution is preferred for fixation in experiments involving RNA extraction because paraformaldehyde cross links with RNA and thus makes its recovery difficult [14,17,18,30]. After fixation the tissues are embedded either in paraffin at room temperature or in an optimum cutting temperature (OCT) medium at −20 °C (cryosectioning). While OCT-embedded samples are sectioned through a cryomicrotome at −20 °C, the paraffin-embedded samples are sectioned at room temperature. Cryosectioning is often preferred over sectioning of paraffin-embedded samples because processing the tissue at a low temperature reduces the activity of RNases and increase yield [1,23]. Nevertheless keeping the RNA intact during the various steps of the cryosectioning for LCM poses a challenge and entails fixing and processing tissues. For example, plant cells contain big vacuoles for water storage, which may form crystals because of fast freezing of samples during cryosectioning leading to the destruction of cellular structures. Similarly, it is challenging to maintain the histological preservation during cryosectioning so that the target cells can be distinguished from others cell types. Therefore a number of tissue fixation and embedding procedures have been developed to ensure optimised handling of tissues with different physical rigidity [4].
Nakazono et al. [23] were the first to report application of LCM in plant samples by isolating the epidermis and vascular tissue cells of maize plants, which were subsequently used in microarray studies. Soon after, Kerk et al. [17] presented an RNA isolation technique using the paraffin-embedded sections of a variety of plant tissues. Further the LCM procedure has been applied to isolate syncytial material from soybean roots infected with cyst nematode H. glycines [15,18,19]. These authors applied paraffin embedding to process the syncytial samples and showed that a number of genes are differentially regulated in these plants upon nematode infection. But the utility of this protocol for nematode-infected Arabidopsis roots remains questionable as it may lead to poor quality RNA extraction. LCM was also used to capture giant cells and isolate RNA induced by root-knot nematodes in Arabidopsis, rice and tomato during different stages of infection [1,3,7,16,24,26]. However no protocol describing the isolation of syncytial cells from Arabidopsis have been described so far. In the present study we optimised the LCM procedure for the isolation of syncytial cells from Arabidopsis roots in such a manner that their morphology can be preserved and high-quality RNA can be isolated for downstream applications such as microarray analyses.

Results
To assess the potential of a previously described giant cell RNA isolation protocol in Arabidopsis for its utility for syncytial samples, we grew Arabidopsis plants in KNOP medium and infected them with J2 nematodes of H. schachtii. Root segments containing syncytia were cut at 5 days post inoculation (dpi). The infected root segments were subsequently fixed in EAA (3:1) and processed for LCM as described previously [1,23,24]. However, RNA was heavily degraded in these samples and was unusable for downstream applications (data not shown).
To identify the crucial step in which the RNA was degraded, we hand-dissected root segments containing syncytia at 5 dpi and isolated RNA at three different steps ( Table 1). The subsequent quality analysis revealed that the RNA was already degraded before embedding in OCT. Hence sections mounted on the slides already contained deteriorated RNA (Fig. 1a, b). Conversely the RNA isolated from syncytial tissues that were fixed but not treated for cryoprotection produced high-quality RNA (Fig. 1c). We concluded that the cryoprotection procedure needs to be modified to obtain good quality RNA.

Modification of cryoprotection steps yielded good quality RNA
To investigate whether RNA degradation might have occurred because of multiple incubations in different sucrose concentrations (10 % for 3 h, 15 % for 3 h and 34 % overnight) during cryoprotection; we modified the tissue preparation procedure by reducing the sucrose incubation steps. For this, root segments containing infected tissues were cut and fixed in EAA solution followed by a direct incubation in 15 % sucrose solution for 3 h (low concentration sucrose-treated samples). These samples were then left overnight in 34 % sucrose solution. Alternatively fixed tissues were directly incubated in 34 % for 5 h (high concentration sucrose-treated samples). Subsequently, tissues from low-and high-concentration sucrose-treated samples were embedded in OCT, cryosectioned and used for RNA isolation. While the RNA from low-concentration sucrose-treated tissues was heavily degraded (Fig. 2a), the sections from high-concentration sucrose-treated tissues showed high-quality RNA (Fig. 2b). Therefore these samples were further processed (mounting on slides, and removing OCT) to isolate syncytial sections through LCM. Subsequent RNA isolation showed a slight degradation in quality of RNA; however, both the quantity and quality of RNA was still reasonable to perform downstream applications (Fig. 2c). We concluded that incubating fixed samples directly in a 34 % sucrose solution for 5 h improves the RNA quality considerably.

Incubation in higher sucrose concentrations affected tissue morphology
Although incubating fixed samples directly in 34 % sucrose for 5 h resulted in improved RNA quality, the tissue morphology was poor (Fig. 3a, b). We reasoned that this might be due to shorter and direct incubation in high concentration sucrose, which may lead to sudden loss of tissue pressure. Therefore we increased the vacuum infiltration of samples in 34 % sucrose from 15 to 45 min followed by an overnight incubation at 4 °C. As a consequence, tissue morphology was improved to normal (Fig. 3c). The RNA quality of the samples was not affected by increasing the incubation time.
Poly-l-lysine-coated glass slides strongly adhere to micro-sections Different types of glass slides with diverse features are available. In this study, four types of slides were tested to obtain a strong tissue adherence: (a) polyethylene teraphthalate (PET)-membrane-coated slides, (b) polyethylene napthalate (PEN)-membrane-coated slides, (c) Superfrost ® positively charged slides, and (d) polyl-lysine-coated glass slides. We mounted 10 µm sections on PET-and PEN-membrane-coated glass slides and washed them with PBS buffer and 70 % ethanol to remove the OCT medium. The complete removal of the OCT medium is necessary for laser cutting. However during washing, all sections were washed away from the slides. We pre-treated the slides with UV light for 30 min to 4 h to enhance adherence, but the majority of the sections was still washed off during the washing step. Next we used Superfrost ® positively-charged glass slides, on which tissue retention was greatly improved, while the use of poly-l-lysine-coated glass slides proved robust in holding the tissues during the washing step. Hence, in all subsequent experiments, we used poly-l-lysine-coated glass slides, and the laser capture machine was also calibrated to glass slides.

In vitro amplification of cDNA
To assess the quality of RNA isolated through our protocol for downstream applications, cDNA was synthesised from 5-days-old syncytial using NuGEN's Ovation Pico WTA System according to the manufacturers' instructions. Subsequent analysis showed that enough high quality cDNA was amplified (Fig. 4a-c). The amplified cDNA was used to perform further steps of hybridization with the GeneChip ® Arabidopsis ATH1 Genome (Affymetrix UK Ltd). The data analysis showed that amplified cDNA was suitable for microarray experiments (data not shown). Whereas the detailed results from microarray will be presented elsewhere, we analysed the expression of six different genes (BGLU28, PGIP1, PDF1.4, WRKY76, Xylanase, and ß tubulin4) in 5-days-old syncytial samples by RT-PCR (see methods for details). These genes were selected based on intensity of expression in syncytium in our microarray data (data not shown). Under optimized PCR conditions, five out of six genes  (Fig. 5). Nevertheless, these results confirm that RNA isolated through LCM protocol as described in this manuscript can be used to study gene

Discussion
After the successful use of LCM on animal and human tissues, it has