Laser microdissection on Norway spruce bark tissue: A suitable protocolfor subsequent real-time reverse transcription–polymerase chainreaction (RT-PCR) analysis

N. LUCHI1, P. CAPRETTI1, C. G. FOSSDAL2, M. PAZZAGLI3, & P. PINZANI3

1Dipartimento Biotecnologie Agrarie, Sez. Protezione delle Piante, Universita degli Studi di Firenze, Firenze, Italy, 2The

Norwegian Forest and Landscape Institute, As, Norway, and 3Dipartimento Fisiopatologia Clinica, Sez. Biochimica Clinica,

Universita degli Studi di Firenze, Firenze, Italy

AbstractThe development of new tools able to select specific plant tissue is crucial for gene expression studies. During the last years,the use of laser microdissection, mainly tested on herbaceous plant tissue, has been found to be a useful technique for thesepurposes. This method is poorly tested on woody species, and so far no studies of gene expression have been applied onforest trees. For this reason the present work proposes the optimization of a functional protocol using laser microdissectionpressure catapulting (LMPC) and real-time reverse transcription–polymerase chain reaction (RT-PCR) in bark stem tissueof Norway spruce (Picea abies). Bark tissue fragments were collected from Norway spruce trees and sliced with a cryostat.RNA was extracted from both whole cross-sections and microdissected bark cells. The feasibility of the method wasconfirmed by the amplification of the a-tubulin, an endogenous gene of P. abies, with efficiency comparable to that obtainedfrom non-microdissected tissue. The proposed protocol, here adapted for bark tissue of woody species, represents a usefultool in a wide range of hosts that, unlike herbaceous plants, have scarcely been considered up to now.

Keywords: a-Tubulin gene, bark, laser microdissection pressure catapulting, Picea abies, RT-PCR, woody plants

Abbreviations: LAM, laser-assisted microdissection; LMPC, laser microdissection pressure catapulting; RT-PCR,reverse transcription–polymerase chain reaction.

Introduction

During the last years laser-assisted microdissection

(LAM) has been devised to dissect morphologically

homogeneous cells from microscopic tissue sections

(Day et al. 2005; Pinzani et al. 2006), facilitating the

quest for cell-specific information (Kehr 2003;

Brandt 2005). LAM includes ‘‘laser microdissection

pressure catapulting’’ (LMPC) – (P.A.L.M. Micro-

laser Technologies AG, Bernried, Germany). This

system uses an inverted microscope and a nitrogen

laser for the selection, microdissection, and subse-

quent recovery of a single cell or a small group of

morphologically defined cells by catapulting them

into a collection cap using the same laser source. The

collected cells can then be used for RNA and DNA

extraction and for downstream applications such as

expression studies or gene copy number evaluation

using real-time quantitative methods (Pinzani et al.

2006, 2008).

LAM has been widely used, principally in medi-

cine, where, combined with real-time RT-PCR

quantification, it makes possible expression studies

of tumor cells buried among other cells in biopsy

tissues (Gelmini et al. 2006; Morrogh et al. 2007).

However, devising useful protocols for the micro-

dissection of tissue for clinical studies has turned out

to be a laborious undertaking, the major difficulties

being in the preparation of the specimens for LAM,

and even more in retrieving and amplifying the

nucleic acids isolated from the microportions of fixed

tissue (Pinzani et al. 2006).

LAM has been used much less frequently in

plants, where it is mostly applied to herbaceous

Correspondence: N. Luchi, Dipartimento Biotecnologie Agrarie, Sezione Protezione delle Piante, Universita degli Studi di Firenze, Piazzale delle Cascine 28,

50144 Firenze, Italia. Tel: þ39 0553288275. Fax: þ39 0553288273. Email: nicola.luchi@unifi.it or nicola.luchi@libero.it

Plant Biosystems, 2011; 1–7, iFirst article

ISSN 1126-3504 print/ISSN 1724-5575 online ª 2011 Societa Botanica Italiana

DOI: 10.1080/11263504.2010.505017

individuals, since woody tissues such as those of the

stems of trees, are more difficult to prepare and cut

with a laser. Among studies on herbaceous plants,

some have dealt with gene expression in rice and

maize (Zhang et al. 2007; Hobo et al. 2008) some

with transcriptomic and proteomic analysis in maize

and Arabidopsis thaliana (L.) Heynh. (Dembinsky

et al. 2007; Day et al. 2008), and others with the

metabolites and chemical compounds in A. thaliana,

nettle, and alfalfa (Schad et al. 2005; Angeles et al.

2006; Nakashima et al. 2008). There are also some

studies on gene expression involved in plant–patho-

gen interactions, such as the interaction of nema-

todes on soybean (Klink et al. 2010), the powdery

mildew on Arabidopsis (Chandran et al. 2010), and

that of the anthracnose stalk rot fungus on maize

(Tang et al. 2006).

Up to now few studies, carried out on olive and

citrus, have examined laser microdissection to study

gene expression of woody plants (Corpas et al. 2006;

Agustı et al. 2009). However these studies examined

leaves and not woody tissue. A protocol for the LAM

of a conifer tree such as Norway spruce, which has

thicker lignified tissues than the herbaceous plants

mentioned above, represents a new challenge in

RNA studies. The aim of this study was to optimize

LMPC combined with real-time RT-PCR in order to

detect a-tubulin gene expression in Norway spruce

bark tissue. Effectiveness of the method on micro-

dissected tissue has been tested with particular

attention to the efficiency of amplification in whole

cross-sections on a-tubulin transcript amount. The

a-tubulin gene has previously been used as an

endogenous reference transcript in gene expression

studies in Norway spruce because its expression

levels are relatively stable (Johnsen et al. 2005;

Yakovlev et al. 2006) and little affected by treatment

such as stem wounding and inoculation with fungal

pathogens (Hietala et al. 2004; Johnk et al. 2005).

Here it was used as an example of a constitutively

expressed gene.

Materials and methods

Plant material and preparation of sections

Three healthy 30-year-old Norway spruce (Picea

abies (L.) Karst.) trees were selected in the Mon-

tesenario forest (Florence, Italy). Rectangular strips

of living bark (ca. 26 5 cm, 1-cm thick) were

excised with a scalpel from the base of the trees

down to the sapwood surface. Samples were im-

mediately frozen in liquid nitrogen and stored at

7808C until use. The samples were divided into

smaller plugs (ca. 56 56 5 mm) and then sliced to

a thickness of 10, 15, and 30 mm with a cryostat

(Frigomat, Jung, Germany) at 7208C to obtain an

optimal thickness. Three cross-sections were cut for

each tree, floated on diethylpyrocarbonate (DEPC)-

treated H2O, and then stretched on PALM1

membrane slides (1-mm glass microscope slides

coated with a 1.35-mm poly-ethylene-naphthalene

membrane). The slides were washed two times in

DEPC water and once in RNaseZap (Ambion,

Cambridgeshire, UK), and then placed for 30 min

in a UV light chamber, in order to remove the

nucleases and increase their adherence to the

membrane. Sections were then stained in 0.1%

toluidine-O-blue and dehydrated in an ethanol series

(containing DEPC water) at 70%, 95%, and 100%

for 2 min for each step.

Laser microdissection pressure catapulting

Bark cells were microdissected from sliced sections

using the PALM1 Microbeam System (P.A.L.M.

Microlaser Technologies) which employs laser pres-

sure catapulting and cold ablation by a UV laser

source (337 nm nitrogen laser), with an inverted

microscope (Carl Zeiss, Milan, Italy). The laser was

used with pulse energy higher than 270 mJ, 3 ns pulse

duration, and a cutting speed of 30 pulses s71. The

area selected for cutting was visualized on a

computer screen, and a mass of cells was dissected

with the laser beam at 406 magnification and

directly catapulted into a cap tube (Eppendorf,

Hamburg, Germany) containing 20 ml of buffer

RLT (Qiagen, Milan, Italy). The tubes were then

centrifuged at full speed for 5 s before RNA

extraction.

In order to estimate the number of cells present in

the microdissected samples, the area of the indivi-

dual cells was measured with the microscope system,

and expressed as mm2. This information was then

used to calculate the number of cells present in the

microdissected samples.

RNA extraction and reverse transcription (RT) reaction

Total RNA was extracted both from the whole cross-

sections and from microdissected Norway spruce

tissue, following the protocol of the RNeasy Micro

kit Total RNA isolation from microdissected cryo-

sections (Qiagen). To ascertain whether the tolui-

dine-O-Blue stain interfered with the RNA isolation

yield, RNA was extracted from both stained and

unstained whole cross-sections.

The RNA was extracted with an RNeasy MinElute

spin column, DNase treated, and eluted in 14 ml

of RNAse-free water. To each sample, 0.4 U ml71 of

RNAse inhibitor was added. The concentration of

the extracted RNA was measured using a Nano-

drop1 ND-1000 spectrophotometer (NanoDrop

Technologies, Wilmington, DE, USA).

2 N. Luchi et al.

Eight microliters of the total extracted RNA was

reverse-transcribed in a final volume of 20 ml

containing: 106 TaqMan RT Buffer, 5.5 mM

MgCl2, 2.5 mM of each dNTP, 1.25 U ml71

Multiscribe RT, and 1.25 mM random hexamers.

All RT reagents were purchased from Applied

Biosystems (Foster City, CA, USA). The RT

reaction was carried out on a Bio-Rad Thermal

Cycler System (Bio-Rad, Milan, Italy) kept at 258Cfor 10 min, at 488C for 30 min for reverse transcrip-

tion (RT), and at 958C for 5 min to denature the

enzyme.

Real-time PCR and the a-tubulin assay

The level of the Norway spruce a-tubulin gene

transcript used as an endogenous reference for real-

time studies (Hietala et al. 2004; Johnk et al. 2005)

was assayed using SYBR green chemistry. Each

reaction was performed in a total volume of 25 ml

containing 12.5 ml SYBR1 Green PCR Master Mix

(Applied Biosystems), 1.25 mM of each of the reverse

(50-AAGTTGTTGGCGGCGTCTT-30) and for-

ward (50-GGCATACCGGCAGCTCTTC-30) a-

tubulin primers (described in Hietala et al. 2004),

and 5 ml of cDNA. Real-time PCR was performed on

an ABI PRISM 7700 (Applied Biosystems) as

follows: a first cycle at 958C for 10 min; and 50

cycles at 958C for 15 s and at 608C for 1 min. Each

sample was measured in triplicate. Three wells

without cDNA were used as the no-template

controls, representing a negative reference.

Four 10-fold serial dilutions of RNA extracted

from Norway spruce bark tissue were used as a

standard to obtain a calibration curve to calculate the

amount of a-tubulin transcript in the unknown

microdissected tissues. The standard curve ranged

from 25 ng to 25 pg of total RNA extracted from

Norway spruce. The choice of a bulk tissue RNA

extract as a standard has the advantage to exhibit

features very close to those of the unknown samples

and thus to amplify with comparable efficiency.

PCR efficiency of sample amplification (whole and

microdissected cross-sections) was calculated by

using the program LinRegPCR 11.0, for the analysis

of quantitative RT-PCR data (Ruijter et al. 2009).

For each sample the program determines a window-

of-linearity and then uses linear regression analysis to

fit a straight line through the PCR data set. From the

slope of this line the PCR efficiency of each

individual sample is calculated (Ruijter et al. 2009).

In order to evaluate the effectiveness of LMPC

method, the estimated total RNA amount, based on

the transcript level of a-tubulin, was normalized per

area of extracted tissue (pg mm72). Quantification

was carried out for whole sections (stained and

unstained) and for the LMPC samples.

Results

Cross-sections in Norway spruce bark

The size of the cryosections of Norway spruce bark

tissue was adjusted to a thickness of 15 mm. This was

the optimal thickness that allowed the target cells to

be preserved without any solvent or cryoprotection,

leaving the single cortical cells structurally intact

before LMPC. In some cases cross-sections thicker

than 15 mm (i.e. at 30 mm) failed to stretch out on

the PEN membrane of the slide used in LMPC. In

other cases, sections thinner than 15 mm (at 10 mm)

were extremely fragile. Even at a thickness of 15 mm

there were some cell damage and cracking in the

sections. This was particularly evident for the resin

duct cells.

Laser pressure catapulting microdissection

Analyzed single bark cell had an average area of

2148.65 mm2, (n¼ 20; SD¼ 629.87), ranging from

1147 to 3294 mm2 (Figure 1a) as evaluated using the

PALM1 Microbeam System. Three distinct micro-

areas, measuring about 100,000 mm2, were micro-

dissected from each tissue slice. In some cases the

whole microdissected area (corresponding to ap-

proximately 50 bark cells) could not be catapulted in

one go, so it was decided to divide the total area to be

dissected into microareas of about 40,000 mm2 each.

For each of these microareas the tissue was selected

(Figure 1b), cut twice by a laser microbeam (Figure

1c), and catapulted into the lid of a collection tube

(Figure 1d).

RNA isolation and a-tubulin detection by real-time PCR

Total RNA was isolated with values ranging between

51.5 ng ml71 (mean value in whole cross-sections)

and 6.5 ng ml71 (mean value in microdissected tissue

that correspond *0.13 ng total RNA per cell). We

also performed preliminary extraction tests on bark

cells using Trizol, following the protocol described

by Kerk et al. (2003), which gave comparable results

(data not shown), but we decided to use the Qiagen

kit because it was less time-consuming.

LMPC did not affect the amount of the detected a-

tubulin transcript with respect to the whole cross-

sections. All the samples gave positive results with

the real-time RT-PCR assay for a-tubulin (Figure

2a). In view of the fact that in RT and subsequent

amplification the volumes of RNA and cDNA

employed are portions of the total volume, the

average Ct value obtained for the microdissected

sample corresponded to the amount of transcript

extracted from five to six bark cells. The standard

curve used to quantify the a-tubulin transcript

exhibited a slope¼7 3.321; a Y-intercept¼ 40.44,

Laser microdissection on Norway spruce 3

and R2¼ 0.998. PCR efficiency calculated on the

kinetic obtained from microdissected samples

(1.930; R¼ 0.999) did not differ significantly

(p4 0.05) from those obtained in whole cross-

sections, both stained (1.933; R¼ 0.999) and un-

stained (1.974; R¼ 0.999) (Figure 2b and c).

Similarly, the amount of a-tubulin mRNA per area

did not differ significantly (p4 0.05) between

LMPC and the whole cross-sections, whether treated

with toluidine or not (Figure 3).

Discussion

In the present work it was demonstrated that LMPC

could be successfully used to obtain RNA for real-

time RT-PCR quantification in a woody plant such as

Norway spruce. So far, two methods have mainly

been employed to prepare sections for laser micro-

dissection in plants (mostly in herbaceous plants):

paraffin-embedded sectioning and cryosectioning

(Inada & Wildermuth 2005; Scanlon et al. 2009).

The first gives excellent histological resolution, but

sample preparation (fixation, dehydratation, and

embedding) requires a large number of steps and

this may lead to a partial loss of macromolecules such

as RNA, due to fixation, diffusion and oxidative

processes. The second method often gives a lower

resolution than the previous one from a histological

point of view but is more effective when the main

objective is RNA or DNA isolation (Kerk et al. 2003).

In herbaceous plants, cryosectioning typically

causes ice crystals to form inside the vacuoles,

damaging part of the sample (Huang et al. 2002);

for this reason, to obtain optimal histological

sections, immersion in sucrose has been suggested

(Casson et al. 2005). However, Schad et al. (2005),

in a study of the vascular bundles of Arabidopsis

found that good cryosections could also be obtained

without any immersion in sucrose. This simpler

procedure improved preservation of the cellular

structure and minimized loss of metabolites and

macromolecules such as RNA and proteins.

Taking this last study as our starting point, we

developed a protocol for the microdissection of

Figure 1. Laser microdissection pressure catapulting (LMPC) in Norway spruce cortical cells. (a) Selection and measure of the area

(expressed in mm2) of each single cell. (b) Cells, encircled on the computer screen, can be cut by LMPC. (c) After cutting, an empty space

was observed on the slide and microdissected tissue was catapulted (d) into the cap of an Eppendorf tube. Magnitude (a) and (d):640; (b)

and (c):610.

4 N. Luchi et al.

cortical cells of Norway spruce without using any

solvents or cryoprotectants. In our case the rather

rigid structure of Norway spruce tissue limited frost

damage. Nevertheless, there were problems in

obtaining sections thin enough to get good morpho-

logical definition and to ensure that the laser would

efficiently cut through the cell walls of the bark.

Li et al. (2007) used laser microdissection for

microchemical analyses in stone cells of Norway

spruce. The thickness of their cross-sections, ob-

tained after embedding and cryosectioning, was

30 mm, and laser microdissection was performed by

using a Leica AS LMD System (Leica, Bensheim,

Germany), where the microdissected samples were

collected by gravity. In our experience a slice

thickness of 30 mm did not allow a proper visualiza-

tion of the tissue morphology nor to get precise and

efficient laser cutting. For this reason a thickness of

15 mm was chosen as the best compromise between

Figure 2. Selection of a-tubulin amplification plots showing the comparison among whole stained cross-sections and microdissected tissue.

(a) PCR efficiency was calculated for each single plot by LinRegPCR 11.0 (Ruijter et al. 2009). An example of LinRegPCR analysis for a

whole cross-section (b) and microdissected tissue (c) is reported.

Figure 3. Real-time RT-PCR on RNA extracted from

Norway spruce cortex. Mean (+SD) of a-tubulin expression

(pg total RNA mm72) in microdissected area and whole

sections (stained and unstained) (n, cross-sections per tree¼ 3;

n, trees¼ 3).

Laser microdissection on Norway spruce 5

histological integrity and laser cutting suitability,

even if the cell walls on account of their lignification

sometimes still resisted cutting.

In addition, problems during laser cutting may be

due to an inadequately performed tissue preparation,

such as the dehydration. This step is very important

because it avoids the formation of small water bub-

bles, which may prevent tissue adhesion to the slide,

making laser microdissection impossible (Pinzani

et al. 2006). In our case the ethanol series used was

suitable for Norway spruce bark tissue.

The use of stain is particularly important in LAM

because it facilitates identification of the various cell

types and brings the retrieved sample into better

view. Our results showed that toluidine, already used

in conifer tissue of Pinus nigra Arn. (Luchi et al.

2005), did not significantly affect the yield of total

RNA, as demonstrated by the results obtained with

real-time RT-PCR from whole tissue sections

(Figure 3).

In the present study, in which LMPC was

combined with RNA extraction, it was also necessary

to find a balance between reasonable histological

resolution and macromolecule extraction. The use of

cryosections was helpful to detect specific cells for

harvesting and allowed the macromolecules to be

isolated. RNA was successfully extracted from both

whole sections and the microdissected areas, by a

spin-column based extraction method (RNeasy

Micro kit) commonly used for animal tissues

(Pinzani et al. 2006). The effectiveness of the

extraction method has been demonstrated, as re-

ported in Figure 3, where the amount of a-tubulin

expression did not differ between microdissected and

whole samples. This result has also been confirmed

after PCR efficiency analysis that showed no

differences not only between these two kinds of

samples (Figure 2b and c), but also between whole

stained and unstained cross-section, showing that

toluidine did not affect the PCR efficiency.

The microdissection protocol combined with real

time RT-PCR in Norway spruce bark samples

demonstrated the suitability of the method in heavily

lignified plant tissue. Consequently, the objective of

the present study was fully reached in devising a

protocol for laser microdissection, followed by RNA

quantitative determination for forest trees. In the last

few years the use of laser microdissection has

revealed itself to be a useful tool to study gene

expression in plant–microbe interactions (Balestrini

& Bonfante 2008; Gomez & Harrison 2009;

Chandran et al. 2010). The development of this

protocol for the bark stem tissue of forest trees

represents an important new way to study gene

modulation at the level of specific cells during the

interaction of cells of Norway spruce with patho-

genic microorganisms.

Acknowledgements

This study was carried out by grants from the

University of Florence ‘Fondi di Ateneo’. We grate-

fully acknowledge the Section of Plant Biology (DiBA,

University of Florence) for cryostat sectioning.

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Laser microdissection on Norway spruce 7