the field of gene expression analsysis has evolved dramatically in recent years . with a basis in microarray technology and the ongoing transition into next - generation sequencing technologies ,
the microarray technology provides a way of obtaining huge quantities of genome and transcriptome data ; it has developed from relatively small - scale experiments using in - house platforms and protocols to more robust studies using what are essentially genome - wide commercially manufactured arrays .
today , several commercial and academic platforms exist , with different array manufacturing and sample preparation approaches .
a common criticism of the field of global gene expression analysis has been its lack of standardised experimental protocols and well - defined reference studies comparing different platforms and procedures .
to address these issues , the microarray quality control consortium ( maqc ) performed a study where relative gene expression measurements , using one and two - colour platforms , were compared within and between platforms , as well as with taqman real - time pcr data     [ 13 ] .
the reference data set , based on standardised commercially available rna sources , was made publicly available , enabling researchers to benchmark new procedures and platforms .
one overall purpose for developing new protocols for global gene expression analysis is to improve the quality of the results produced . during recent years
the quality of microarrays has been further improved in terms of content and fabrication procedures     [ 46 ] , ensuring limited slide - to - slide and batch - to - batch variability .
equally important is sample preparation , during which several of the steps may introduce additional variability into the experiment . to minimise variation within an experiment ,
an experienced technician   and a good laboratory protocol are required . in general , the statistical power increases as the variance in the experiment decreases , increasing the likelihood that , for example , differentially expressed genes and subtle differences between samples will be detected . in order to achieve these goals , we present a method where all the major steps in a typical cdna library preparation protocol are automated using a robotic workstation capable of handling superparamagnetic beads .
our procedure performs cdna synthesis , purification and subsequent labelling using nhs - modified fluorophores , and is capable of handling up to 48 samples per instrument in parallel .
studies have indicated that automation of sample preparation for single - colour microarray experiments can reduce variation and increase throughput     [ 7 , 8 ] .
we present a similar approach , to benchmark our protocol against the freely available data from maqc to validate our results , and compare the automated approach to our own manual procedure as well as to several academic and commercial platforms .
several methods for the purification of nucleic acids are available today , including ethanol precipitation and spin - column - based methods .
these methods generally require manual input , thereby limiting the   throughput of the experiment and increasing variability . in order to reduce human input and increase reproducibility , an automated protocol for cdna synthesis ,
labeling , and purification has been developed using a dedicated instrument capable of handling superparamagnetic beads .
the procedure is outlined in figure 1 , where the two purification steps are highlighted . for a full run of 48 samples ,
the current protocol takes about five hours , with most of the time being incubation steps ( e.g. , cdna synthesis takes two hours ) .
performing 24 similar reactions manually can be done in approximately the same time , but manual handling introduces variation that can be avoided using the automated approach .
the automated protocol outperform the manual procedure when it comes to within - experiment correlation between replicates .
an overview of the automated approach for total rna followed by cdna synthesis and labelling in illustrated in figure 1 .
this procedure consists of several critical steps that were optimised , including evaporation from open wells , investigation of bead capacity , and proper clean - up from free fluorophores . in conclusion , we show that an automated approach for cdna synthesis performs better than a similar manual procedure when interrogated using dna microarrays .
we believe that this conclusion is valid for other readouts platforms as well , including rna - sequencing     [ 911 ] . in order to increase the yield from the purification steps
briefly , following the first elution , the beads are returned to the supernatant , enabling    capture of any residual cdna .
this approach increases the yield from each purification step by approximately 15% , which has a significant impact on the overall yield of labelled cdna , given that the process makes use of two purification steps ( data not shown ) .
only small amounts of beads are necessary for a high yield : approximately 1.8 g of labelled cdna can be purified using as little as 20 g of beads if there are two captures during each purification step ( figure 2 ) . to facilitate complete capture
, we use 150 l of beads , thus purifying approximately 5 g of labelled cdna .
precipitation is carried out in an ethanol / teg buffer , after which the bead pellet is washed five times in 80% ethanol .
a large number of washes are necessary to remove unincorporated fluorophores , but does not affect the yield of sample cdna ( data not shown ) .
elution can easily be performed in a low salt buffer , such as water . in order to test the performance and reproducibility of the automated process
, we took advantage of the well - documented model system used by the maqc consortium     [ 13 ] .
the aim of the macq study was to create a freely - available large reference data set and to compare multiple transcription profiling platforms using a standard set of rna samples .
we chose a subset of these platforms for our analysis . for within - experiment comparisons , the two - colour experiments , nci 3 and 4 ( national cancer institute ) ,
these platforms use the human probe set version 3 from operon , which is also used on the slides in our study .
we used the same sources of rna , total human reference rna ( stratagene ) , and firstchoice   human brain reference total rna ( ambion ) to evaluate and compare the automated protocol with the manual procedure for cdna synthesis and labelling prior to microarray analysis .
ten replicates were run in two separate automated experiments and were compared with ten replicates prepared according to the manual procedure .
the differential gene expression between the two sources of rna was measured in these experiments as outlined for the previous macq experiments .
figure 3 shows the overall results by comparing the correlation between the m - values ( log2(sample a / sample b ) ) from our experiments ( kthauto and kthman ) and m - values from different experiments in previous studies    [ 2 , 3 ] , including only two - colour platforms .
the samples from our two automated experiments cluster together closely and cluster less closely to the manual samples . within the  automated  cluster , two samples ( kthauto_b2 and kthauto_a4 )
are set slightly apart from the other samples but are close to each other ; they are still , however , within the same super - cluster ( blue cluster , figure 3(a ) ) . in both of these samples , the cdna synthesis and labelling procedure yielded about two thirds the amount of labelled cdna compared to the other samples ( this was due to manual handling - related issues ) , while the degree of labelling was about the same ( data not shown ) .
this may indicate that the total amount of hybridised material could play an important role in creating reproducible results .
the median spearman correlation of all available m - values between samples labelled using the automated protocol was 0.92 and 0.91 for the two parallel experiments ( kthauto1 and kthauto2 , resp . ) , compared to 0.86 for the manual procedure ( kthman , figure 3(b ) ) indicating an increase in well - to - well reproducibility .
this is in concordance with previous studies     [ 7 , 8 ] . in the wider context
, a higher correlation between replicates means greater statistical ability to detect differentially expressed genes .
when comparing the top 200 differentially expressed genes , 175 ( 87.5% ) are common between kthauto1 and kthauto2 , which can be compared to 155 ( 77.5% ) between   nci3 and nci4 ( supplementary figures   1(a ) and   1(b ) in supplementary material available online at doi:10.1155/2009/396808 ) . in the maqc study ,
after mapping the probes to refseq      and aceview , a complete list of 12091 probes for each platform was created , representing 12091 refseq entries in 12091 entrez genes     [ 3 , 14 ] .
after filtering out genes with stable expression levels across all experiments ( see section 2 ) , we used this list to calculate the spearman 's rank correlation coefficient between mean m - values from each experiment . when compared to other platforms investigated in the maqc project , the data generated using the automated approach cluster together with the two - colour arrays used by nci .
this is to be expected since , as aforementioned , both nci and the arrays used in the automation experiments made use of   the human probe set version 3 from operon . as expected from previous studies     , all one - colour platforms
( ge healthcare , applied biosystems , agilent one - colour , illumina , and affymetrix , see      for more information on these platforms ) cluster together ( figure 4 ) . when comparing the spearman correlation values , the correlation between the kth auto experiments is 0.97 , whereas the highest correlation between the different experiments performed by nci is 0.94 .
a higher correlation between experiments ( lower technical noise ) means that samples can be added later , while introducing relatively little variability to the data .
here , we chose to take advantage of an efficient in - tip magnetic separation system     [ 15 , 16 ] to assess throughput and variance within a gene expression experiment .
there are several approaches available for nucleic   acid purification using paramagnetic beads ; these include the use of streptavidin - coated beads and a biotinylated primer in the reverse transcription step .
however , unless strictly controlled , free biotinylated primers or free biotinylated nucleotides can rapidly saturate the beads , leading to a low purification yield . here
we show that , by precipitating the first - strand cdna on carboxylic acid coated paramagnetic beads , a high yield can be achieved in the purification steps , giving a large quantity of purified product . using this automated procedure ,
thus , automation of cdna library preparation for analysis on microarrays or using massive - scale sequencing      [ 911 ] leads to decreased variance and greater statistical power to detect for example differentially expresssed genes or alternative splicing patterns .
we describe an automated platform that can be used to increase the robustness of the overall performance of cdna library preparation , including target labelling .
the maqc study indicated two types of variation : array content and array performance , the latter relating to the manual variation associated with performing the experiments .
we show that this variation can be minimised by using an automated procedure , employing a standard microtiter plate .
the number of samples that can be run in parallel can also be increased using this protocol from one up to 48 .
a single row in the microtiter plate ( 1 to 12 samples ) takes approximately 4 hours and 40 minutes and the time increase for running four rows ( 48 samples ) is marginal . some modifications , other than changing the purification method , were necessary when automating the process .
these were mainly due to reactions associated with the microtiter wells , which have no lids , so that evaporation occurs and is pronounced at elevated temperatures .
the amount of evaporated water at a given temperature , however , is relatively constant . during the initial total rna denaturing step , about 3.5 l of water evaporates , and this
prior to optimisation , the amount of cdna obtained from the first strand synthesis on the workstation was approximately 20% less than from synthesis in closed tubes .
this effect was completely eradicated when water was added at regular intervals during the cdna synthesis ( data not shown ) .
the adjusted volume of the samples was determined empirically . to investigate the performance of our automated procedure , we chose dna microarrays because of the access to a well established reference set of rna samples and the standardized statistical procedure to address and demonstrate technical variation .
in general , well - to - well correlation increased when compared to a manual protocol .
correlation between different experiments ( i.e. , the same experiment performed at different days ) , we noted a higher correlation using our automated procedure when compared to the procedure carried out by nci .
our results indicate that automation of sample preparation improves technical reproducibility , and should be general indifferent of the platform for readout , dna microarrays or rna - sequencing .
in all experiments , total human reference rna ( stratagene ) ( sample a ) and firstchoice human brain reference total rna ( ambion ) ( sample b ) were used .
the two rna samples were labelled as described below , and subsequently hybridised to microarrays . in each experiment , a total of 10 microarrays was used . on five of these , sample a labelled with cy3 and sample b labeled   with cy5 were hybridised . on the remaining five slides ,
the dye setting was reversed , giving a dye - balanced direct design , corresponding to the two - colour microarray experiments that were carried out within the maqc study . using the optimised protocol , we performed two experiments following the automated approach and one following our manual procedure .
for the automated sample preparation , the process from total rna to purified labelled cdna was performed on a magnatrix 1200 ( magnetic biosolutions , sweden ) robotic workstation .
this workstation is equipped with a heating block and a system for in - tip magnetic separation . for initial cooling in the instrument
the protocol takes approximately 4 hours and 40 minutes for a single microtiter row of 12 samples , and only slightly longer for the double amount of samples .
twenty micrograms of total rna was primed with 5 g of random hexamers ( invitrogen , usa ) in a total volume of 22 l depc - treated sterile deionised water .
after denaturing the rna for 10 minutes at 70c , the mixture was cooled in a pcr - cooler ( eppendorf ) in the instrument , maintaining 0c in the wells for 5 minutes prior to starting the first strand cdna synthesis .
after this step , approximately 3.5 l of liquid had evaporated , resulting in a total volume of about 18.5 l .
a volume of 11.5 l of a reverse transcription master mix was then added , setting the reaction composition to 1x first - strand buffer ( invitrogen ) , 0.01 mm dtt ( invitrogen ) , 0.4 mm aminoally l - modified dutp ( biotium ) , 0.1 mm dttp ( sigma - aldrich ) , 0.5 mm datp , dctp , and dgtp ( sigma - aldrich ) and 400 units of superscript iii reverse transcriptase ( invitrogen ) . an initial incubation of 10 minutes at room temperature ( approximately 22c ) was followed by two hours at 46c . during the incubation , 2.5 l depc - treated sterile deionised water was added every 15 minutes to compensate for evaporation .
the cdna synthesis reaction was stopped by the addition of 3 l 0.2 m edta after which rna was hydrolysed by the addition of 5 l 1 m naoh together with incubation at 70c for 15 minutes .
ph neutralisation was achieved by the addition of 15 l 1 m hepes , ph 7.0 , after the hydrolysis step .
prior to purification , the storage buffer was removed from 150 g of dynabeads myone carboxylic acid ( invitrogen ) by magnetic separation .
the beads were resuspended in the neutralised cdna synthesis mixture , after which three volumes ( 150 l ) of binding buffer , containing 80% ethanol and 6.7% teg , were added . after a single 10-minute incubation at room temperature ,
the beads were collected from the supernatant and washed five times in 45 l 80% ethanol .
elution was carried out by resuspending the beads in 10 l sterile deionised water and mixing for 1 minute . to increase the yield , collected beads were resuspended in 5 l of sterile deionised water and returned to the supernatant for a second capture .
the incubation , washing and elution steps were then repeated as described above , resulting in a final volume of 20 l . in order to set the ph to facilitate coupling to fluorophores , 2 l 1 m nahco3 , ph 9.0 , was added to the eluted cdna , after which the mixture was transferred to a 5 l aliquot containing one tenth of the contents of mono - functional nhs - ester cy3 or cy5 dye tubes ( ge healthcare ) dissolved in dmso ( sigma - aldrich ) . after a single 30-minute incubation at room temperature , 2 l of 1 m hepes , ph 7.0 ,
was added to neutralise the mixture prior to purification of fluorophore - coupled cdna using carboxylic acid coated paramagnetic beads as described above .
briefly , the pre - cdna synthesis incubation was performed at   25c instead of room temperature , and the purification steps were carried out using the minelute cleanup system ( qiagen ) according to the manufacturer 's recommendations .
differences include changing the recommended washing buffer before labelling the cdna to 80% ethanol and the elution buffer to sterile deionised water .
moreover , the neutralisation step was carried out using   5 l 1 m hcl .
the arrays used were provided by the kth microarray center ( http://www.ktharray.se/ ) , and consisted of the human genome oligo set version 3.0 ( operon ) printed in 30% dmso onto ultragaps slides ( corning ) .
after printing , the slides were uv cross - linked at 150 mj / cm .
the slides were prehybridised for 30 minutes at 42c in a prehybridisation solution consisting of 5x ssc , 0.1% sds ( sigma - aldrich ) and 1% bsa ( sigma - aldrich ) to avoid unspecific hybridisation to the glass surface .
the slides were subsequently washed in water and isopropanol ( sigma - aldrich ) and dried using a slide centrifuge .
the two samples were dye - balanced to make sure that equal amounts of dye were hybridised in each channel , then they were pooled and denatured ( 3 minutes at 95c ) in a hybridisation mixture containing 50% formamide ( sigma - aldrich ) , 5x ssc and 0.1% sds ( sigma - aldrich ) , 20 g human cot-1 dna ( invitrogen ) , and 20 g yeast trna ( invitrogen ) .
the 65-l hybridisation mixture was then cooled on ice for 1 minute and applied under a cover slip ( erie scientific company ) , placed on top of the printed array ; it was then hybridised for 24 hours at 42c in a water bath .
following hybridisation , the slides were washed with increasing stringency using 2x ssc and 0.1% sds at 42c , followed by 0.1x ssc and 0.1% sds at room temperature and finally by five repeated washes with 0.1x ssc at room temperature .
the arrays were scanned at 10-m resolution using an agilent g2565ba scanner ( agilent technologies , usa ) , with the photo multiplier tube set to 100% for each laser .
the images thus acquired were analysed using the irregular gridding algorithm in genepix pro 5.1 ( axon ) and the resulting data were imported into the r environment for statistical computing and visualisation .
the raw data were extracted from the median foreground intensity for both channels and subsequently filtered on the basis of flags ( features either not found by the image software , or marked as bad spots ) , a low signal compared to the local background and saturated signals .
data normalisation was carried   out using a block - wise lowess approach , included in the aroma package   and a per - feature mean log2-ratio ( m - value ) across all slides was calculated using the limma software package .
the data was compared with relevant parts of the maqc data set , using clustering and correlation analysis tools in the kth software package .
mean fold change and p - values for each platform were calculated , and all contrasts set up between all platforms using the limma software package .
the files containing raw data were made publicly available in the arrayexpress repository     , with experiment number e - tabm-749 .
for intraplatform comparisons , a subset of platforms with the same probe set ( operon human probe set version 3 ) and experimental design from maqc was used .
features were mapped using the oligonucleotide i d , which is a unique identifier for each oligonucleotide sequence . for interplatform comparisons ,
the m - values for 12091 genes common to the platforms were extracted , as well as a subset of 906 genes with available taqman real - time pcr - data ; these were compared to all the microarray platforms within maqc .
genes for which the interquartile range was lower than 0.5 were removed from the analysis , in order to reduce background noise from genes with stable expressions levels across all tested platforms .
in addition to hybridising the labelled cdna samples to microarrays , the binding capacity of the carboxylic acid - coated paramagnetic beads was measured .
the number of washes after binding the cdna was optimised , after which elution was carried out and cdna and fluorophore concentrations were determined using a nanodrop nd-1000 instrument ( nanodrop ) .
 
 background . 
 several technologies , such as in - depth sequencing and microarrays , enable large - scale interrogation of genomes and transcriptomes . in this study , we asses reproducibility and throughput by moving all laboratory procedures to a robotic workstation , capable of handling superparamagnetic beads . 
 here , we describe a fully automated procedure for cdna synthesis and labelling for microarrays , where the purification steps prior to and after labelling are based on precipitation of dna on carboxylic acid - coated paramagnetic beads . 
 results . 
 the fully automated procedure allows for samples arrayed on a microtiter plate to be processed in parallel without manual intervention and ensuring high reproducibility . 
 we compare our results to a manual sample preparation procedure and , in addition , use a comprehensive reference dataset to show that the protocol described performs better than similar manual procedures . conclusions . 
 we demonstrate , in an automated gene expression microarray experiment , a reduced variance between replicates , resulting in an increase in the statistical power to detect differentially expressed genes , thus allowing smaller differences between samples to be identified . 
 this protocol can with minor modifications be used to create cdna libraries for other applications such as in - depth analysis using next - generation sequencing technologies . 
1. Introduction
2. Results
3. Discussion
4. Materials and Methods