traditionally , to obtain enough material for global transcript 
 analysis , whole organs or tissues have been used . however , by 
 starting from a mixture of cells or tissues , key changes occurring 
 in just one of the cell types may be obscured . to avoid this , 

approaches have been developed that allow high - resolution tissue 
 sampling or enrichment for specific cell types . 
 in some cases , such as pollen grains or epidermal cells ,
however , 
 most cell types are embedded and require tissue digestion or 
 microdissection to negate surrounding cell layers .
laser 
 microdissection ( lm ) is an important method for obtaining 
 individual tissues , cells , and even organelles for biochemical 
 analysis . originally developed for isolating cancerous cells from 
 normal tissue   , lm is beginning to be used in plant 
 biology ( reviewed by ) .
compared to manual 
 microdissection , modern laser - based systems are easy to use , 
 highly reproducible , and can avoid direct contact with the 
 biological sample .
lm samples have been used successfully 
 to obtain dna , rna , proteins , and metabolites from a range of 
 plant species and 
 tissue types [ 57 ] .
one disadvantage of isolating specific cell types is the 
 relatively large amount of effort required to isolate a very small 
 amount of material . in the cases of protein and metabolite 
 analysis , microdissection is still a very inefficient approach for 
 global analysis [ 6 , 7 ] .
however , rna can be amplified ; a 
 process that can be carried out using a variety of methods 
 . the most common method of amplification is based on 
 in vitro transcription ( ivt ) .
this linear 
 amplification method involves producing double - stranded cdna with 
 a t7 priming sequence at the 3 end .
this template is then used 
 by t7 rna polymerase to generate copies of the cdna template . 
 
compared to ivt - based methods , the exponential nature of pcr - based 
 approaches enables much greater yields per round of amplification 
 .
this means that far less starting material can be used 
 to obtain enough target for high - throughput analysis , with just 
 one round of amplification . for both ivt- and pcr - based methods , 
 multiple rounds of amplification
can be used to increase the 
 yields when starting with very small amounts of rna . 

hybrid methods have also been developed that use pcr and ivt 
 amplifications , in an attempt to exploit the advantages of both 
 [ 11 , 12 ] .
rna amplification from very small amounts of rna by ivt or pcr has 
 several drawbacks . reducing the starting amount of rna to nanogram 
 levels
however , since biases tend to be 
 systematic , accurate expression data can be obtained if 
 appropriate amplified controls are used .
for example , 
 when comparing expression changes between samples , it is essential 
 that all samples are similarly amplified .
arabidopsis is being used as a model plant to study many 
 aspects of seed biology ; however , a major drawback is the very 
 small size of its seeds .
this causes numerous technical 
 difficulties , especially when studying processes of early seed 
 development .
the endosperm appears to play several important roles 
 during seed growth and development . in many plant 
 species ,
including arabidopsis , the triploid primary 
 endosperm nucleus undergoes several rounds of free - nuclear 
 division , growing rapidly as a syncytium . towards the end of this 
 proliferative phase ,
the micropylar endosperm surrounds the 
 embryo , the peripheral domain lines the wall of the 
 developing embryo sac , and the chalazal domain develops 
 adjacent to the vascular connection with the seed 
 parent .
the early proliferation of the endosperm is associated 
 with final seed size and the alteration of the rate and duration 
 of cell division in the endosperm has been proposed as a biotech 
 strategy for altering seed size 
 [ 15 , 16 ] .
seed with severely 
 defective endosperm can not complete development which suggests a 
 role for endosperm in supporting the formation and growth of the 
 embryo .
lm provides an ideal tool for analyzing gene 
 expression changes in specific cell types during the early stages 
 of arabidopsis seed development .
the key to the success 
 of such studies is the ability to isolate and amplify rna from a 
 small amount of laser - dissected tissue . here , we present protocols 
 for the isolation of rna from different seed compartments of 
 developing arabidopsis seed and highlight important 
 technical issues encountered when amplifying from small amounts of 
 total rna for array analysis .
the protocols developed in this 
 study were used to identify a list of approximately 2 700 genes 
 that are expressed in proliferating endosperm 4 dap .
arabidopsis thaliana plants were grown under lights in a 
 growth room with 16-hour photoperiod and at 20c . to 
 produce seed of a known developmental stage ,
flowers were 
 emasculated with fine forceps and manual pollinations were carried 
 out two days after . 

multiple siliques corresponding to early and late stages of seed 
 development were harvested into eppendorf tubes and snap frozen in 
 liquid nitrogen .
siliques from the early sample were added to the 
 late sample at a ratio of 1 to 10 ( based on fresh weight ) to 
 produce a 90% late sample . early and
90% late tissues were 
 ground to a fine powder in liquid nitrogen using a pestle and 
 mortar .
total rna was isolated from the samples using concert plant rna 
 purification reagent ( invitrogen , carlsbad , calif , usa ) 
 following the manufacturers ' large - scale extraction protocol . 

aliquots of these samples were further purified using qiagen 
 rneasy columns and the manufacturers ' optional on column dnase 
 step was carried out . 
 
siliques were harvested 4 days after pollination into precooled 
 ( 4c ) farmers ' fixative ( 3 : 1 mixture of 
 ethanol : acetic acid ) . to enhance penetration of 
 the fixative , the ends of the siliques
samples were kept for 3 - 4 hours 
 on an orbital platform at 4c .
the siliques were 
 transferred into sample cassettes that were then submerged in 
 absolute ethanol and were microwaved at 60c for 
 10 minutes
microwave 
 treatment was repeated in two changes of isopropylalcohol at 
 70c for 10 minutes and a third change at 
 74c for 10 minutes .
cassettes were then transferred 
 to molten wax and placed in a heated vacuum chamber .
siliques were 
 released into molten wax and multiple siliques were aligned in 
 single wax blocks and stored at 4c with desiccant for up 
 to two weeks before being sectioned using a rotary microtome 
 ( leica microsystems , wetzelar , germany ) to produce 
 7 m sections .
the sections were floated on distilled water and then 30% ethanol 
 prior to attachment to superfrost plus slides ( bdh / merck , 
 damstadt , germany ) .
slides were allowed to dry for 2 hours before 
 storage with desiccant at 4c . 

slides were dehydrated in xylene for 5 minutes and allowed to 
 air dry in a flow hood for 510 minutes prior to lcm using 
 the arcturus lm200 pixcell ii system .
this system enables specific 
 regions of the tissue sample to be targeted microscopically 
 through a plastic cap coated with a special thermoplastic film . 

the laser was then used to melt the film onto the targeted areas 
 using a spot size of 7.5 m , power setting of 100 mw , 
 and pulse durations of 1 - 2 milliseconds , depending on the ease of 
 capture from each specific seed .
once cemented to the film , 
 targeted tissues were removed from the rest of the section by 
 lifting the cap vertically .
a post - it note was 
 used to remove the contaminating cells ( 3 m , st paul , mn , usa ) .
the remaining 
 tissue was sometimes scraped from the glass slide using a sterile 
 flat - ended spatula , and was subsequently processed using 
 equivalent protocols to the lcm material . 

total rna was obtained from embryo , seed coat , syncytial 
 endosperm , and the tissue scrape samples using the picopure 
 rna isolation kit ( arcturus , oxnard , ca , usa ) with 
 optional on - column dnase step ( qiagen , valencia , ca , 
 usa ) .
the purified total rna was quantified using the ribogreen 
 rna quantification kit ( invitrogen ) according to the 
 manufacturers ' instructions . 

aliquots of 30 g total rna were reverse transcribed and 
 labelled using the superscript indirect labelling kit ( invitrogen ) . 

aliquots of 50 ng total rna were subjected to reverse 
 transcription using superscript iii reverse transcriptase 
 ( invitrogen ) and amplified dsdna was prepared following the 
 methods described by petalidis et al .  
, with the exception 
 that the entire cdna sample was used in the amplification 
 reaction .
a test pcr is essential to avoid overcycling of the 
 amplification reaction ; however , this effectively doubles the 
 amount of total rna required to carry out rna amplification . to 
 determine the optimal number of cycles required for generating 
 products during the exponential phase of the pcr reaction , pcr 
 products from parallel runs
were visualised after 15 , 18 , 21 , and 
 24 cycles on a 1.2% agarose gel .
when the yield of pcr product 
 stops increasing with more cycles , the reaction has reached its 
 plateau .
the optimal number of cycles for the amplification is one 
 cycle fewer than is needed to reach the plateau ; in our case 20 
 cycles .
amino - allyl dntps were incorporated by klenow and the free 
 cy dyes ( amersham biosciences , little chalfont , uk ) were 
 coupled to the target dsdna as described in the superscript 
 indirect labelling kit manual ( invitrogen ) .
ivt - based amplifications were carried out using the message amp ii 
 amino - allyl arna kit ( ambion inc .
where appropriate , 
 amino - allyl utps were incorporated into the ivt reaction and were 
 coupled to cy dyes ( amersham biosciences ) following the kits 
 recommendations .
all labelled arna targets were fragmented prior 
 to hybridization using ambion fragmentation reagent following the 
 manufacturers ' protocol ( ambion ) .
the rna or arna was primed with random hexamers and 
 first - strand cdna was synthesized using superscript iii 
 ( invitrogen ) according to the manufacturers ' 
 instructions .
the amplification conditions for conventional pcr were as 
 follows : 2 minutes at 94c ; 2535 cycles of 30 
 seconds at 94c , 30 seconds at 58 , 30 seconds at 
 72c ; and 68c for 2 minutes .
the primers 
 for fwa , mea , and ap2 are described by 
 kinoshita et al . 

real - time qrt - pcr was carried out using reagents from the 
 lightcycler faststart dna masterplus sybr green i kit 
 ( roche , penzberg , germany ) in 20 l volumes using a lightcycler 2.0 
 ( roche ) .
the amplification conditions for qpcr were denat : 
 95c for 10 minutes ; cycling 94c for 5 
 seconds , 58c for 17 seconds , 72c for 10 
 seconds ( single acquire ) ; melt : 95c for 0 second , 
 55c for 20 seconds , 95c for 0 second with ramp 
 0.2c / s ( continuous acquire ) ; cool : 40c for 20 
 seconds .
the differential expression 
 observed across the 17 spots per slide representing actin-2 in our 
 normalized data was 1.004 indicating equivalent expression in both 
 types of biological sample used to query the arrays and used for 
 qrt - pcr .
reaction products were confirmed by melting curve 
 analysis and by running out the product on a 1.2% agarose gel . 

primer sets for qrt - pcr validation experiments were designed to 
 span at least one intron so we could easily observe pcr products 
 generated from genomic dna contamination . for each primer set , a 
 no - template water control was also used . 

the primers used for qrt - pcr of fwa and mea are 
 described by kinoshita et al . 

[ 19 , 20 ] 
 and the fis2 
 primers were fis2-rt - r 5-agatctcctggcgactaac-3 , and 
 fis2-rt - f 5-tattagcgaacgcctgagac-3. actin primers were q - rt 
 actin_f 5-cgctctttctttccaagctcat-3 and q - rt actin_r 
 5-tcctgcaaatccagccttc-3. tt8 primers were tt8-qrt - f 
 5-ctgatcttcatattgaatcaaccca-3 and tt8-qrt - r 
 5-gtgtgacatgagaagtgttgttac-3. fus3 primers were fus3-qrt - f 
 5-tcatggtctgcagctaggtga-3 and fus3-qrt - r 
 5-tacttcttcttcttccgatgcttt-3. at5g16780 primers were 
 cas5g - qrt - f 5-gtgggtttgcgactgttg-3 and cas5g - qrt - r 
 5-gagtttccgggctctgatt-3. at1g48630 primers were cas1g - qrt - f 
 5-aagtctgttgttgaggatttgaag-3 and cas1g - qrt - r 
 5-ttccatctgcactccagtt-3. 
  prewash and bsa prehybridization : slides were prewashed 
 in 0.2% sds for 2 minutes and then washed twice in milliq 
 ( mq ) water .
the slides were then placed in a 50 ml falcon tube 
 containing fresh mq water and were incubated in a water bath for 
 20 minutes at 50c .
slides were then prehybridized in 
 buffer containing 4x ssc , 0.5% sds and 5% w / v bovine 
 serum albumin ( bsa , sigma ) at 42c for 45 minutes . 

slides were then washed 5 times with mq water at room temperature 
 to remove bsa residue .
hybridization : hybridizations were carried out under 
 lifterslips ( erie scientific , portsmouth , nh , usa ) placed over the region of 
 the slide containing the array . a mix of slidehyb buffer 1 
 ( ambion ) and the labelled probes
the arrays were then placed in a humidified 
 hybridization chamber ( corning , new york , ny , usa ) and were hybridized over 
 night in a 55c water bath .
posthybridization : the slides were removed from the 
 hybridization chamber and were washed at 55c for 8 
 minutes with agitation in 50 ml falcon tubes containing 2x 
 ssc , 0.1% sds .
the slides were then moved to new 50 ml falcon 
 tubes and were washed with constant agitation in 1x ssc , 0.1% 
 sds at room temperature for further 6 minutes .
this procedure 
 was repeated with a solution containing 0.5x ssc and then 0.1x ssc 
 before the slides were dried by centrifugation at 5800 rpm .
scanning : slide scanning was carried out using an axon 
 4000 b dual - color scanner running genepix 4.1 software ( axon 
 instruments / molecular devices corporation , sunnydale , ca , usa ) . 
 
genepix files were normalized by implementing elements of the 
 snomad gene analysis tools 
 ( http://pevsnerlab.kennedykrieger.org/snomadinput.html ) at a 
 local level using an splus script .
this involved global mean 
 normalization , local mean normalization across the array surface , 
 and local mean normalization across the element signal intensity . 

data from high and low scans were normalized 
 independently . when calculating correlations , the number of 
 differentially expressed transcripts and the direction of change , 
 the saturated spot data was replaced with data from the low 
 scan .
any spots that had intensity values less than 3x 
 the mean background in both channels or were only represented in a 
 single replicate were discarded from this analysis .
genes that had 
 a signal above the trim level in only one channel remained in the 
 analysis but had a value of 100 intensity units interpolated for 
 the low signal to reduce the effect of very low intensity features 
 on expression ratios .
data was 
 normalized without interpolation for use with genes that had very 
 low signal , for example , fis2 .
when interpreting the data 
 with regard to movement of intensity values within the population 
 or loss of signal based on probe design , we only considered the 
 high intensity scan and the data was not trimmed based on 
 intensity .
the microarray data has been deposited at ncbi geo 
 ( http://www.ncbi.nlm.nih.gov/geo ) accession number gse6703 .
laser capture microdissection ( lcm ) is a form of lm that relies on 
 the adhesion of only the targeted cells to a thermoplastic film 
 attached to a plastic cap .
we developed tissue 
 preparation protocols for lcm of immature arabidopsis 
 seed using farmer 's fixative based on recommendations of kerk 
 et al . .
other groups have successfully used acetone or 
 microwave fixation in the absence of chemical fixative to carry 
 out transcript analysis on plant material 
 [ 21 , 22 ] . however , 
 our preliminary studies indicated that farmer 's fixative gives the 
 most consistent penetration of the fixative into 
 arabidopsis silique tissues and enables good histology 
 and yields of total rna . using farmer 's fixative ,
our tissue 
 fixation period ( 3 - 4 hours ) was shorter than protocols using 
 acetone ( overnight ) .
we also used the heat generated by a 
 microwave to aid the diffusion of subsequent solvent washes and 
 the infusion of wax under vacuum , further reducing the processing 
 time .
this enabled silique material to be processed from harvest 
 to wax block within 9 hours .
figure 1(a ) shows examples 
 of lcm isolations of seed coat , endosperm , and embryo tissue from 
 developing siliques .
lcm removed the majority of the target tissue 
 and only the desired cells were visible on the lcm cap 
 ( figure 1(a ) ) .
fixed samples were aligned in the wax so 
 that approximately six siliques were within the footprint of an 
 lcm cap .
three serial sections were mounted onto a single 
 glass slide , enabling the caps to be rapidly repositioned over 
 each serial section in turn if target tissue was limiting ( as was 
 the case for embryo samples ) . as well as allowing sufficient histological resolution , the 
 tissue - processing regime must be conducive to subsequent 
 extraction of rna of sufficient quality and in amounts suitable 
 for meaningful analysis .
we used the arcturus picopure rna 
 isolation kit with an on - column dnase treatment to isolate total 
 rna . in an alteration to the kit protocol , we routinely peeled the 
 thermoplastic film from lcm caps using fine forceps and submerged 
 these in extraction buffer .
this allowed multiple caps to be 
 extracted in the same tube . by combining two peels containing 
 isolated material from multiple seeds
, we were able to obtain 
 2060 ng of total rna per extraction , depending on the seed 
 compartment targeted , for example , approximately 50 
 endosperm fragments were obtained per peel enabling 100 fragments 
 to be used for rna extraction ( figure 1(b ) ) .
the rna 
 yields were quantified using the ribogreen rna quantification kit 
 ( invitrogen ) . 
 as with most studies of this kind , we assessed the quality of the 
 extracted rna by generating cdna templates from unamplified 
 samples and conducting rt - pcr  
 [ 5 , 21 , 23 , 24 ] .
we also used 
 small aliquots of arna produced from one round of ivt 
 amplification for similar analysis ( 1015% of total product ) . 

the cdna yield from the arna increased our effective yield of 
 experimental material and would be sufficient to carry out several 
 hundred tissue - specific pcr reactions .
our lab is interested in identifying genes that are important 
 during early endosperm development . to identify transcripts 
 expressed specifically in the endosperm
, we needed to ensure that 
 the other seed compartment samples had minimal endosperm 
 contamination and our endosperm sample was suitably enriched .
to 
 investigate this , we carried out rt - pcr on lcm - derived samples .
as 
 shown in figure 1(c ) , ap2 was amplified from all 
 samples using conventional rt - pcr , consistent with its reported 
 expression throughout the early seed , and as expected 
 mea expression was relatively strong in the endosperm and 
 weak in the embryo ( globular to mid - heart ) .
we used arna 
 from a single round of ivt amplification to assess the enrichment 
 of endosperm expressed transcripts by real - time qpcr .
we 
 calculated the levels of expression ( relative to actin ) of two 
 endosperm - specific genes , fwa and fis2 , and 
 mea from both lcm - derived endosperm cdna ( 4 dap ) and a 
 similarly amplified tissue scrape cdna .
the scrape sample was 
 derived from the tissues remaining on the slide after 
 microdissection of the endosperm .
not all seed tissues enabled 
 unambiguous identification and unimpeded access to endosperm 
 tissue , thus the tissue scrape retained a proportion of endosperm 
 material .
results indicated a strong enrichment for endosperm 
 ( figure 2 ) , such that the endosperm - specific genes 
 fis2 and fwa reported 9.5-fold and 8-fold 
 enrichment , respectively .
mea reported a 3.5-fold 
 enrichment that reflects its simultaneous expression in the embryo 
 [ 25 , 26 ] .
taken together , our pcr analysis indicated that the rna obtained 
 using lcm generated expression data that was consistent with the 
 expression of known marker genes and that the cell types were well 
 separated .
our aim was to identify which standalone amplification protocol 
 performed best in our lab when the starting amount of total rna 
 was limited to 50 ng . while other studies have compared 
 amplification protocols , few start with such low amounts of total 
 rna   [ 10 , 13 ] . in addition , most of these studies use 
 ultra - high - quality rna bought commercially or isolated from cell 
 lines [ 2733 ] . 

since the quality of extracted 
 rna is very often tissue - dependent and will affect the 
 amplification efficiency , we decided to use total rna extracted 
 from whole siliques ; better representing tissues targeted by lcm . 

we set out to compare two basic methods of rna amplification , 
 based on ivt and pcr .
preliminary amplifications from lcm prepared 
 samples indicated that two rounds of ivt or a single round of pcr 
 were required to generate enough arna or cdna , respectively , for 
 hybridization to long oligonucleotide arrays ( data not shown ) . 
 

rna was isolated from siliques during early ( 25 dap ) and late 
 ( 610 dap ) stages of development . to help identifying major 
 alterations in the array data due to our target preparation 
 protocols ,
the late stage rna sample was spiked with 10% early 
 stage sample ( 90% late ) . in the absence of compression ,
the 
 signals from the genes only expressed in the early sample should 
 not be more than 10-fold higher than those obtained from the late 
 sample .
this design should result in the 
 unamplified ma plots ( plots of differential expression against 
 intensity ) having a distinctive asymmetrical shape . 
 

our study compared unamplified samples and three rna amplification 
 regimes ; one - round ivt , two - round ivt , and a pcr - based method .
the 
 unamplified treatments used 30 g of total rna followed by 
 conventional labelling and hybridization .
the one - round ivt 
 amplifications were made from 5 g of total rna ( a 
 standard labelling procedure in many microarray experiments ) .
the 
 two - round ivt and pcr regimes started from 50 ng of total rna 
 and would therefore be appropriate for use with lcm samples .
the 
 unamplified treatment involved duplicate hybridizations of the 
 90%-late sample against our early sample . for each of the 
 amplification regimes ,
a single amplification of the 90%-late 
 sample was hybridized against two independent amplifications of 
 our early sample . in all hybridizations
the early and 90%-late 
 samples were consistently labelled with cy5 and cy3 , respectively . 

this consistent labelling strategy for all 8 slides reduced 
 variation between the microarrays due to dye bias and enabled us 
 to focus on the variation arising from duplicate amplifications of 
 a single early silique sample . the rna yields after amplification are given in 
 table 1 .
we aimed to generate 1020 g of arna 
 using ivt and 15 g of cdna using pcr ; such that we 
 could comfortably perform duplicate hybridizations from each 
 sample .
when starting with 50 ng of rna , the desired amount of 
 arna / cdna was obtained after either two rounds of ivt 
 ( 103 g arna ) or a single round of pcr amplification 
 ( 10 g ) , respectively ( table 1 ) .
ivt - based 
 amplification on our lm samples generated arna with a similar 
 efficiency to that obtained from a conventional tissue sample 
 ( table 1 ) . 

the reproducibility and fidelity of the amplification methods were 
 assessed by microarray analysis using long oligonucleotide arrays 
 based on the operon v1.0 arabidopsis set .
the emergence 
 of extremely high signal for some array elements post 
 amplification ( hotspots ) has been described in the literature 
   and was countered in this study by carrying out both 
 high and low power scans for each hybridization .
data from high 
 and low scans were normalized independently and the saturated spot 
 data replaced with data from the low scan .
this enabled the 
 highest possible number of data points to be included in our 
 analysis and negated any issues due to alteration of signal spread 
 due to different amplification regimes . in instances where data 
 from the combined scan set might introduce artefacts into our 
 analysis , we considered only data from the high power scans . 
 to assess the reproducibility of our methods , we plotted the 
 duplicate data obtained for each target preparation regime 
 ( figure 3(a ) ) .
the between - replicate reproducibility of 
 the unamplified samples was high with a pearson correlation of 
 r = .95 .
the single - round ivt amplification gave an even higher 
 between - replicate correlation ( r = .98 ) , a phenomenon that has 
 been reported by others [ 35 , 36 ] .
the extreme amplifications 
 also showed high between - replicate correlations of r = .8 and 
 r = .9 for pcr and 2ivt , respectively . 

puskas et al .    also 
 found a higher correlation with ivt than with pcr , when the two 
 amplifications were compared directly .
although our pcr and 2ivt 
 correlations were somewhat lower than the more conventional target 
 preparation strategies , they are consistent with other studies 
 using rna samples diluted down to tens of nanograms ( r = .87.94 ; 
 [ 38 , 39 ] ) . in summary , amplification of 50 ng of rna by either two rounds 
 of ivt or pcr produced sufficient product for reproducible microarray hybridizations . to determine if the different amplification methods fundamentally 
 altered the global representation of the transcript populations , 
 we first considered variations in the ma plots .
these plots graph 
 intensity on the x - axis versus log2 of the differential 
 expression on the y - axis .  
our experimental design generated the 
 expected asymmetrical form when the unamplified 
 data were plotted in this way ( figure 4 ) .
all 
 amplification regimes deviated from this pattern and appeared to 
 compress the differential signals reported in the unamplified 
 dataset .
the magnitude of compression was ( from lowest to highest ) 
 ivt , 2ivt , and pcr .
the compression comprised a more reduced 
 differential in the direction of the 90%-late sample than that 
 observed in the early sample .
this suggests that highly 
 differential values were more affected than moderate fold changes . 

this was also evident in the correlation plots drawn between the 
 amplified data and the unamplified data , where the most 
 differential signals fell away from the r = 1 line 
 ( figure 3(b ) ) .
the amplification regimes also reported 
 a more consistent spread of intensity values 
 ( figure 4 ) . to assess the affects of amplification on relative 
 transcript abundance , we calculated the total intensity ( sum of 
 red and green signals ) for each gene   within each 
 treatment .
these values were placed in rank order and were split 
 into five groups based on intensity .
the transcripts contained 
 within each intensity bin were then tracked back to see which 
 intensity groups the transcript originated from in the unamplified 
 samples .
amplification caused alterations in the relative 
 intensity of a subset of genes ; an effect that    was 
 most pronounced with extreme ivt- or pcr - based 
 amplifications ( figure 5 ) .
pcr was the worst affected , 
 with many transcripts moving into widely different intensity 
 groups . 

our amplified data gave good positive correlations 
 to the unamplified samples ( r = .62.83 ) and independent studies 
 describe similar values [ 40 , 41 ] .
many studies that assess 
 fidelity of amplification have been carried out using affymetrix 
 genechip technology [ 10 , 13 ] .
these studies tend to give 
 higher correlations between amplified samples , than those obtained 
 here using academic printed slides , a situation that is perhaps 
 partly attributable to issues of manufacture .
however , genechip 
 technology can exclude up to 50% of the genes detected by long 
 oligonucleotide - based arrays , based on their low signal intensity . 

furthermore , in the majority of genechip 
 studies , the conventional target preparation method used is a mild 
 form of ivt based - amplification .
if we compare our two - round ivt 
 amplification to our ivt labelled sample our correlation increases 
 from a pearson correlation of r = .78 to .9 .
conversely , our pcr 
 amplified dataset had a lower correlation to the ivt labelled 
 sample than it did to the unamplified dataset .
other aspects of target preparation can also 
 introduce sources of variation that can affect the correlation 
 between datasets .
reverse transcriptases can vary in their ability 
 to yield useful amounts of cdna from limiting amounts of rna 
   and in their ability to generate cdna that largely 
 maintains the length of the original transcript , an important 
 issue when using oligonucleotide - based microarrays ( see later ) . 

comparison of dna hybridizations ( unamplified and pcr ) , with 
 fragmented arna hybridizations ( ivt ) , may also introduce variation 
 due to different hybridization kinetics . 
 
 despite obvious alterations in the observed signal from our 
 hybridizations after amplification , we wanted to assess whether 
 the direction of differential expression was consistent .
the genes 
 that reported a two - fold differential expression in the 
 unamplified treatments were tracked in the amplified data .
it 
 became apparent that while many genes reported an altered level of 
 differential expression , the majority of these genes gave a 
 consistent direction of change , irrespective of the method used or 
 extreme nature of the amplifications ( figure 6 ) . 

considerable variation in fold change has been observed in 
 microarray data sets , compared to real - time pcr 
 [ 4547 ] . 

often microarray data changes are compressed and this phenomenon 
 is more apparent in amplified samples . ignoring or 
 relaxing the fold change cutoff and using statistical methods can 
 to a large extent eliminate problems due to compression .
jenson 
 et al .   found that by dropping the threshold from 2 to 
 1.5 , they got an increased number of differentially expressed 
 genes common to both cdna ( unamplified ) and arna ( amplified ) 
 generated microarray data sets . 
 
overall , our results suggest that while the different 
 amplification methods report variable differential expression for 
 the same gene , they are reasonably reliable at indicating if a 
 gene is up- or down - regulated .
thus , in instances where 
 compression is likely , such as with extreme amplifications , it 
 might be advisable to base interpretation of data mainly on the 
 direction of change .
our experiments were not designed to give precise 
 data about the differential expression of specific genes . 

nevertheless , large fluctuations in the numbers of transcripts 
 reporting two - fold differential expression were apparent , and 
 worthy of comment .
the different amplification regimes reported a 
 sizable percentage ( 6%12% ) of unique differentials 
 ( table 2(a ) ) .
furthermore , the mild ivt amplification 
 reported more genes with a differential expression 
 ( > 2-fold ) than all other treatments , including the 
 unamplified data ( table 2(b ) ) . when we plotted the fold 
 change of the unique ivt and unamplified differentials against 
 their total intensities , the data were similarly distributed to 
 the right of the 3x background intensity cutoff 
 ( figure 7(a ) ) .
however , when we plotted the unique ivt 
 differentials against the intensity values from the unamplified 
 data , it became apparent that the majority of the unique ivt 
 differentials represented transcripts of low intensity in the 
 unamplified dataset ( figure 7(b ) ) .
this indicated that 
 in some instances , amplification can increase the absolute signal 
 intensity allowing more differentials to be identified .
the 
 emergence of such data from low intensity background probably 
 represents a systematic bias in the amplification of specific 
 transcripts .
nevertheless , several studies have verified that 
 differentials specifically identified in amplified samples can be 
 validated by other methods , such as real - time pcr 
 [ 31 , 49 , 50 ] .
taken together , these data suggest that even if rna yields were 
 conducive to conventional cdna hybridizations , the optimum 
 strategy for compiling a list of differentially expressed genes 
 would be several hybridizations using a combination of different 
 target preparation regimes .
the second round of ivt uses a random primer for 
 first - strand cdna synthesis , a process that may lead to truncation 
 of the representative target molecules . to assess the 
 effect of the second - round ivt on our data , each treatment dataset 
 was ranked based on total intensity and the ranks for each probe 

were compared to rankings in the unamplified data.the relative ranking was 
 calculated such that positive or negative changes in rank 
 indicated an increase or decrease in relative signal within the 
 target population , respectively . this change in apparent signal 

was then plotted versus the distance of the corresponding target 
 probe sequence from the 3 end of the annotated target 
 transcript ( figure 8) .
our microarrays were printed 
 using the operon version 1.0 arabidopsis oligonucleotide 
 set . whilst these oligonucleotides were designed to the 3 
 region of genes ,
the plot shown in figure 8 
 indicated that more genes were being lost from our dataset if 
 extreme amplifications were used for target preparation , and that 
 this correlated with the design of some of the microarray oligos 
 being more 5 biased than others .
although not as severe as the 
 target truncation introduced during the random priming of the 
 second round of ivt , pcr amplification also has detrimental 
 affects on target length ( figure 8) .
the template 
 switching method utilized by the pcr regime integrates primer 
 sites into both the 5 and the 3 ends of the representative 
 cdna templates that should negate issues of 3 bias .
however , 
 other studies have also found that pcr can generate products 
 shorter than those obtained from one round of ivt 
 .
this 
 is presumably due to differences in the abilities of different 
 reverse transcriptases to generate full - length cdna . 

the latest version of the operon arabidopsis 
 oligonucleotide set should be less sensitive to target truncation , 
 as it is more 3 optimized .
in addition , not all array types 
 will be as affected by target truncation .
arrays based on printed 
 full - length cdnas , tiling arrays , and conventional affymetrix 
 genechips will have better 3 representation than our arrays . 
 the long oligonucleotide arrays used in this study generally had a 
 single probe representing each transcript .
this is in contrast to 
 affymetrix genechips , which have several probes per transcript 
 that are 3 optimized . in instances where more than one round of 
 ivt are used
generated target populations from lcm harvested embryo cells 
 prepared for genechip analysis by three rounds of ivt - based 
 amplification .
they also found a reduction in 3 representation 
 for some transcripts , but by considering fewer representative 
 probes in their analysis they were able to identify a larger 
 number of embryo - expressed genes . 

we have shown here that the two - round ivt - based amplification 
 provided good between replicate correlations and that it was 
 the extreme amplification method that best represented our 
 unamplified data . however , since this data was obtained using 50 ng of 
 conventionally purified rna rather than rna obtained from a 
 microdissected sample , we investigated whether microarray data 
 produced u   sing lcm - derived rna also gives highly reproducible 
 results .
we used a two - round ivt - based amplification to obtain 
 expression profiles from three replicate endosperm samples 
 microdissected from sections of 4 dap siliques and from the 
 tissues remaining post - microdissection scraped from one of the 
 sample slides .
the tissue scrape arna was used as a universal 
 control for all endosperm hybridizations .
this approach was used 
 to minimize alterations in the measured differential expression 
 due to differences in rna quality . the silique sample ( tissue scrape ) included a 
 proportion of endosperm such that the ma plots for hybridizations 
 between silique tissue scrapes and lm endosperms described 
 an asymmetrical form similar to our previous amplification 
 experiments ( figure 9(c ) ) .
pearson correlations between 
 our replicates were high ( 0.880.93 ) indicating that the 
 microarray data produced using our lm - derived rna generated highly 
 reproducible results .
the lowest correlations were obtained from 
 comparisons between biological replicates that incorporated 
 dye - swaps ( figures 9(a ) and 9(b ) ) .
18 220 unique 
 probes gave signal higher than two - fold background and were 
 represented in at least three of the four hybridizations .
p 
 values were calculated to assess the variation of the signal 
 across the four endosperm hybridizations .
twelve thousand seven 
 hundred and ten ( 12 710 ) probes gave consistent signal with p 
 values of < .05 .
of this list , approximately 5 000 probes 
 allocated to individual loci were differentially expressed in the 
 endosperm sample using a 1.5-fold cutoff or approximately 2700 
 probes if a 2-fold cutoff was used ( listed in the supplementary 
 table 1 available at doi:10.1155/2007/61028 ) . during lcm of endosperm from seed at 4 dap , many individual 
 microdissections were performed .
tissues such as the inner 
 endothelium of the developing seed coat and the developing embryo 
 are immediately adjacent to the endosperm and are the most likely 
 contaminants of our endosperm samples .
few genes have been 
 characterized as being preferentially expressed in the inner 
 endothelium of the seed coat at 4 dap .
however , two such genes tt8 
 and ban 
 [ 55 , 56 ]   gave consistent signal in our array data and 
 were used to assess seed coat contamination ( tt8 was also 
 represented on our slides by two independent probes ) .
both these 
 seed coat markers were excluded from our endosperm expressed list 
 even using a 1.5-fold cutoff , indicating that there is little seed 
 coat contamination of our endosperm lm samples . 
 to screen our data for embryo contamination , we compiled a list of 
 sixteen embryo - expressed genes from the literature . in an lcm 

study that targeted arabidopsis embryos at early stages 
 of development , casson et al .    validated their embryo 
 genechip experiments using a list of embryo - expressed 
 genes .
this study also generated 
 several reporter - gus lines that provided evidence for 
 the expression of several additional genes in early embryos . 

thirteen of the sixteen embryonic markers were excluded from our 
 endosperm - expressed list using a 2-fold cutoff .
of the three 
 embryonic markers present in the endosperm expressed list , 
 lec1 has been reported to be expressed in both the embryo 
 and the endosperm at heart stage by in situ hybridization 
 [ 53 , 57 ]   and there is evidence from an at1g48630 promoter - gus 
 reporter lines that this gene is also expressed in both embryo and 
 endosperm [ 53 , 57 ] . 

the expression of the third embryonic marker , fus3 , in 
 the endosperm at 4 dap is inconsistent with the genes 
 characterization in the current literature . both
fus3 
 promoter - gus    and fus3-gfp translational fusions 
   give strong and highly specific activity in globular 
 embryos .
expression of fus3 in the embryo at heart stage 
 ( approximately 4 dap ) then becomes more restricted to the outer 
 protodermal tissues .
interestingly during the later stages of 
 development , fus3 expression is reported to be strong in 
 the endosperm tissues that form the aleurone layer .
it is 
 possible , therefore , that at 4 dap , fus3 is starting to 
 make the transition from embryonic - specific expression to a more 
 widespread expression pattern .
furthermore , the strong signal from 
 the embryo may obscure a lower more diffuse activity in the 
 endosperm .
appear to be consistent with this 
 hypothesis since the endosperm visible in the heart stage sample 
 ( limited to the embryo surrounding region ) appears to show gus 
 activity .
thus , our endosperm - expressed list does not appear to be 
 contaminated with any genes that are unequivocally expressed 
 specifically in the embryo . to further validate our data , the expression of the two embryonic 
 marker genes not described in the literature as endosperm 
 expressed ( fus3 and at4g48630 )
real - time pcr of the embryonic marker at5g16780 and 
 seed coat marker tt8 were also used to clarify the expression of 
 marker genes close to the 1.5-fold cutoff ( table 3 ) . 
 for the embryonic markers ( fus3 , at1g48630 , and at5g16780 ) 
 real - time pcr gave largely equivalent values to those obtained 
 from the array experiments . for tt8 ,
the differential expression 
 observed by real - time pcr was slightly higher in the endosperm 
 direction than that observed in our array data and took the 
 transcript over the 1.5-fold threshold to a value of 1.66 .
since 
 both tt8 and at5g16780 marker genes gave some evidence of being 
 over 1.5-fold differentially expressed in the endosperm , it was 
 thought prudent to limit the endosperm - expressed list to genes 
 above a 2-fold cutoff ( table 3 ) . to confirm that our approach could detect differential expression 
 of known endosperm - expressed genes , we identified a number of 
 genes from the literature that have been described as 
 endosperm - preferred or endosperm - specific .
tiwari   et al . 
  recently 
 identified a number of candidate genes that gave strong evidence 
 for endosperm - preferred expression .
four of the 
 genes identified as having upstream regions that can drive gus 
 expression during the early stages of endosperm development gave 
 consistent signal in our lm data ( at5g46950 , at5g07210 , at5g39260 , 
 at2g41000 ) .
however , only at5g07210 ( arr21 ) , a two - component 
 response regulator , was identified as having endosperm - preferred 
 expression from our 4 dap data .
interestingly , expression of both at5g46950 and at5g39260 in the 
 atgenexpress genechip developmental series     is only 
 evident in siliques at 3 dap , when the seed contains globular 
 stage embryos , and not in siliques at 4 dap ( heart stage ) .
this 
 suggests that the lm endosperm data is extremely accurate with 
 regard to developmental stage .
further evidence for this is 
 provided in a study that examined the expression of 
 isopentenyltransferases ( ipt ) genes by promoter - gus reporter 
 constructs .
ipt genes catalyze the rate limiting step of 
 cytokinin biosynthesis in arabidospsis , and miyawaki 
 et al
.    found that ipt4 and ipt8 were 
 expressed predominantly in the chalazal endosperm of developing 
 seed .
ipt8 had a slightly more extensive expression 
 pattern in the endosperm and persisted until late heart stage , 
 whereas ipt4 was very specific to the chalazal cyst and 
 the activity in ipt4-gus transformants disappeared prior to heart 
 stage .
the inclusion of ipt8 in our endosperm - preferred 
 list ( and the exclusion of ipt4 ) is consistent with 
 precise sampling of the endosperm at 4 dap when the seeds are at 
 the early to mid heart stages of development .
at2g41000 encodes a dnaj heatshock protein that describes a range 
 of different expression patterns in the literature .
the at2g41000 
 promoter - gus fusion described by tiwari   et al .    was able 
 to drive expression in the endosperm and the embryo sac but was 
 not expressed in pollen .
clarification by semiquantitative rt - pcr 
 was attempted to compare the gus expression pattern with the 
 endogenous transcript but the authors were unable to detect 
 at2g41000 transcript in any tissue .
expression of this gene in 
 online datasets is even more ambiguous with no signal apparent in 
 dissected ovules and 1 dap seed but expression in flower buds , 
 roots , and leaves in the goldberg lab arabidopsis 
 thaliana genechip database 
 ( http://estdb.biology.ucla.edu/genechip ) and only pollen 
 expression being evident in the atgenexpress genechip 
 developmental series .
a study into abscisic acid ( aba ) synthesis has also identified an 
 endosperm - specific 9-cis - epoxycarotenoid ( nced6 ) that 
 cleaves cis - epoxycarotenoids in the first specific step of aba 
 biosynthesis .
nced6 was found to be specific to the 
 endosperm by promoter - gus , promoter - gfp , and in situ 
 hybridization   and is confirmed by the inclusion of nced6 
 in our endosperm expressed list ( table 3 ) .
expression 
 constructs were also made for a second gene , nced9 
 however , despite using over 3 kb of upstream sequence , neither 
 gus nor gfp could be observed in the seed .
this was inconsistent 
 with rt - pcr data from rna obtained from manually dissected seed 
 and in situ hybridization , both of which measure the 
 abundance of the endogenous transcript directly . 

comparisons of endogenous gene expression and reporter activity 
 suggest that the upstream sequences used to drive reporter 
 expression do not necessarily confer expression patterns identical 
 to those of the endogenous gene as assessed by other methods such 
 as in situ hybridization or rt - pcr 
 [ 16 , 58 , 
 63 ] .
another endosperm marker that was consistent with our 
 endosperm - expressed list is suc5 ( table 3 ) . 

the suc5 gene encodes a sucrose transporter that is 
 thought to contribute to the nutrition of the filial tissues 
 during seed development .
examined the 
 expression of the suc5 promoter using both gus and gfp 
 reporter constructs and linked the expression patterns to 
 expression of the endogenous transcript by in situ 
 hybridization studies .
suc5 expression was found to be 
 endosperm - specific , and at 4 dap ( early to mid heart stage ) the 
 expression is changing from localization in the embryo - surrounding 
 region to a more general endosperm expression .
the endosperm - expressed list also contains three components of the 
 fertilization independent seed ( fis ) polycomb group complex ; medea 
 ( mea ) , fis2 , and fie ( table 3 ) .
loss of function 
 mutations of these components of the fis - complex causes autonomous 
 onset of cell division in the central cell without fertilization . 

promoter - gus lines indicate that these three genes are strongly 
 transcribed in the central cell prior to fertilization .
after 
 fertilization , only the maternal alleles of these genes appear to 
 be expressed in the endosperm and persist in the free nuclear 
 endosperm until cellularization [ 65 , 
 72 ] .
as the endosperm 
 cellularizes at approximately 5 dap , gus activity from mea and 
 fis2 reporter constructs decline , whereas fie promoter - gus 
 activity persists and can be observed in both the embryo and the 
 cellularized endosperm .
however , mea expression analysis 
 by in situ hybridization and rt - pcr from manually 
 dissected seed compartments indicate that mea expression also 
 occurs in the embryo at heart stage .
interestingly , the 
 transcripts of mea and fie have relatively low fold 
 changes in our lm endosperm data ( table 3 ) , which is 
 consistent with mea and fie expressions in 
 additional tissues within developing siliques 
 [ 26 ,   65 ] .
the fis complex has been shown to repress the mads - box gene 
 pheres1 ( phe1 ) .
in contrast to the fis 
 genes described above , phe1 is not expressed in the 
 central cell prior to fertilization and appears to be only 
 expressed from the paternal genome after fertilization . 

phe1 promoter - gus reporter lines indicated that 
 phe1 expression is expressed in very early embryos and 
 the endosperm . at heart stage however , phe1 
 expression becomes restricted to the chalazal region of the 
 endosperm , an expression pattern that is consistent with the high 
 differential described by phe1 in our endosperm - expressed 
 list .
another endosperm marker that describes a high differential 
 in our endosperm list is fwa although the function of 
 fwa is unknown , its expression has been characterized 
 using both reporter construct and in situ hybridization 
 and has a similar expression to fis2 .
fwa 
 expression in the endosperm also appears to be solely from the 
 maternal allele .
imprinting in plants has been 
 hypothesized to be an endosperm - specific phenomenon and our list 
 of endosperm - expressed genes includes all confirmed imprinted 
 genes in arabidopsis , that is , phe , 
 mea , fis2 , fwa 
 [ 19 , 26 , 
 62 , 73 , 
 74 ] , 
 and the likely imprinted gene fie 
  ( table 3 ) . in summary ,
our microarray analysis using endosperm - derived rna 
 identified approximately 2 700 genes with putative endosperm 
 expression at 4 dap using a 2-fold cutoff ( supplementary 
 table 1 ) .
the presence of known endosperm marker genes 
 in this gene list indicates that our data is reliable .
with the extensive molecular resources available for 
 arabidopsis and established methods for rna 
 amplification , lm represents an attractive tool capable of 
 providing high - resolution expression analysis and enables novel 
 insights into the cellular processes that occur during early seed 
 development . here , we have established methods for the generation 
 of expression profiles from specific seed tissues and have shown 
 that despite some limitations , global rna amplification from 
 50 ng of endosperm total rna can generate high - quality 
 expression data
. this data should provide a useful resource for 
 laboratories interested in early seed development .
 laser microdissection ( lm ) provides a useful method for isolating specific 
 cells or tissues from biological samples . here , 
 we adapted microdissection 
 protocols to allow high - resolution transcript analysis of different tissues 
 from developing arabidopsis seed . sufficient rna ( 50 ng ) was extracted from 
 endosperm tissue for rt - pcr . however , to obtain enough rna for microarray 
 analyses , it was necessary to amplify the rna . 
 pcr- and ivt - based 
 amplification methods were investigated and several important technical 
 aspects of amplification were identified ( such as target truncation and 
 alterations in signal intensity ) . 
 we found that when starting from only 50 ng of rna , amplification methods based on pcr and ivt produced sufficient 
 product for reliable microarray hybridizations , with two - round ivt giving 
 the best results . 
 microarray analyses , using endosperm - derived rna amplified 
 by two - round ivt , reproducibly identified endosperm enriched marker genes . 
 thus , when combined with rna - amplification protocols , lm is a robust and 
 reliable technique for high - throughput tissue - specific gene expression 
 analysis . 
1. INTRODUCTION
2. MATERIAL AND METHODS
3. RESULTS AND DISCUSSION
4. Conclusions