Optimization of cDNA microarrays procedures using criteria that do not rely on external standards
- Torunn Bruland†1Email author,
- Endre Anderssen†1, 2,
- Berit Doseth1,
- Hallgeir Bergum2,
- Vidar Beisvag1, 2 and
- Astrid Lægreid1, 2
© Bruland et al; licensee BioMed Central Ltd. 2007
Received: 05 March 2007
Accepted: 18 October 2007
Published: 18 October 2007
The measurement of gene expression using microarray technology is a complicated process in which a large number of factors can be varied. Due to the lack of standard calibration samples such as are used in traditional chemical analysis it may be a problem to evaluate whether changes done to the microarray procedure actually improve the identification of truly differentially expressed genes. The purpose of the present work is to report the optimization of several steps in the microarray process both in laboratory practices and in data processing using criteria that do not rely on external standards.
We performed a cDNA microarry experiment including RNA from samples with high expected differential gene expression termed "high contrasts" (rat cell lines AR42J and NRK52E) compared to self-self hybridization, and optimized a pipeline to maximize the number of genes found to be differentially expressed in the "high contrasts" RNA samples by estimating the false discovery rate (FDR) using a null distribution obtained from the self-self experiment. The proposed high-contrast versus self-self method (HCSSM) requires only four microarrays per evaluation. The effects of blocking reagent dose, filtering, and background corrections methodologies were investigated. In our experiments a dose of 250 ng LNA (locked nucleic acid) dT blocker, no background correction and weight based filtering gave the largest number of differentially expressed genes. The choice of background correction method had a stronger impact on the estimated number of differentially expressed genes than the choice of filtering method. Cross platform microarray (Illumina) analysis was used to validate that the increase in the number of differentially expressed genes found by HCSSM was real.
The results show that HCSSM can be a useful and simple approach to optimize microarray procedures without including external standards. Our optimizing method is highly applicable to both long oligo-probe microarrays which have become commonly used for well characterized organisms such as man, mouse and rat, as well as to cDNA microarrays which are still of importance for organisms with incomplete genome sequence information such as many bacteria, plants and fish.
Gene-expression microarrays are widely used for large scale studies in cells, and are emerging as promising tools in clinical diagnosis, with potential impact on the assessment of prognosis and choice of treatment . Microarray data are also increasingly becoming essential for data driven model building in system biology approaches [2, 3]. However, the measurement of gene expression using e.g. cDNA microarray technology is a complex process that involves several steps, which have not yet been fully optimized. In a typical cDNA microarray experiment, total RNA from two biological sources in which gene expression levels are to be compared, is labelled with two different fluorescent dyes and hybridized to an array of spotted cDNA probes. After hybridization and scanning the data are further pre-processed. This often includes image analysis, filtering of data and background subtraction before normalization and transformation of data. Relative gene expression levels are given as ratios of intensities of fluorescent emission from labelled the RNAs'. Many different approaches and methods can be used at each stage of a microarray experiment, and there is still no definitive consensus about which methods to choose (reviewed and discussed in ).
Sample quality, labeling protocol, hybridization conditions, scanning protocols and image acquisition as well as the different stages in the microarray data analysis pipeline can all contribute to the overall uncertainty of the conclusions drawn. There has therefore recently been an emerging focus on the need for universally applicable standards, reference materials and analytic guidelines to assist in the standardization of microarray experiments [4–10]. In order to choose or optimize microarray methods several criteria can be used. The use of external RNA spike-in controls is the most recommended approach for technology assessment and optimization. However, the recommended use of spike-in RNA might not always be feasible. For optimal application several controls and probes must be used and the spike-in controls must be representative of the endogenous RNA with respect to e.g. length, sequence characteristics, melting temperature and cross-hybridization risk . On some arrays suitable spike-in controls are not printed, and printing a sufficient number of spikes can leave insufficient room for the genes of biological interest. Other valuable tools are calibrated reference RNA samples such as those developed in the MAQC project  or mixed tissue RNA samples developed by Thompson et al. [11, 12]. However, it is not always possible to have RNA samples with known differences in the expression level of many genes from the species of interest.
There is a large number of steps in the microarray production, hybridization, scanning and data analysis that have adjustable parameters that may be optimized. We will show how HCSSM can be used to optimize data analysis and hybridization protocols. As mentioned above, the standard microarray data analysis pipeline consists of image analysis, filtering of data, background correction, normalization and identification of differentially expressed genes. Recent studies have focused on the normalization step and evaluated a large number of normalization methods for cDNA microarray data [15, 16]. We have therefore chosen to test the HCSSM on filtering and background correction, as these are fields where there is still considerable debate in the literature [4, 17].
Effects of level of filtration on the number of differentially expressed genes
It is common in microarray analysis to reduce the impact of spots that are malformed or have intensities outside the linear range of the scanner. Such spots are either removed, or weighted down using some measure of spot quality. Filtering has been shown to introduce bias in microarray studies , but has also been shown to significantly reduce variation in self-self hybridizations . However, as far as we know, no work has shown how the bias versus variance trade-off in practice influences the ability to detect differentially expressed genes. Therefore, we tested a number of different filtering methods of increasing complexity to evaluate how they influence the ability to find differentially expressed genes in a study.
Often filtering consists of removing spots with intensities below a (arbitrary) threshold . We have however found no investigations into the effect of such filtering. Several filtering methods based on spot quality statistics have been reported [13, 18, 20]. These quality measures can be used as filters by setting a spot quality threshold, but can also be used as weights to reduce the impact of low quality spots.
Where G and R are the green and red signal intensities, σ R , σ G are the pixel standard deviations in the red and green channel respectively.
The delta term is a small number added to stabilize spots with very small uncertainties.
Effects of background correction on the number of differentially expressed genes
The rationale of background correction is that the e.g. observed green signal intensity (G) is a linear combination of a true signal coming from the labelled RNA (G t ) and a background signal (G b ) so that:
G = G t + G b
More accurate log ratios can then be obtained by correcting the observed signal by subtracting the background. The problem then is to correctly estimate the background signal for each spot in the image analysis phase, and correcting this estimate to satisfy prior criteria e.g. background smoothness and non-negativity of the true signal. Background correction in itself may also destabilize results by increasing variance , and may decrease the ability to detect differentially expressed genes. Therefore, we use the HCSSM to compare the ability to find differentially expressed genes when no background correction is used versus a non negative background correction method .
Effect of choice and dose of dT blockers in cDNA microarray experiments
The HCSS-method can be applied to the laboratory procedures as well as to the data processing steps in microarray research. One major source of bias in cDNA microarrays is cross-hybridization of the labelled RNA to non-target homologous probe sequences on the array [22–24]. A substantial proportion of the non-specific cross-hybridization signal is due to poly(dA)-poly(dT) cross hybridization since poly(dT)-containing molecules produced during labelling of poly(dA) tails of sample RNAs by reverse transcription can bind promiscuously to the poly(dA) stretch of cDNA probe spots. These poly(dA) hybridization signals reduce the ability to detect differentially expressed genes. Thus, minimization of poly(dA) hybridization is a major challenge in cDNA microarray experiments. To reduce the poly(dA) signals it is common practice to add a blocker like synthetic poly(dA) in the probe or poly(dT) in the hybridization mixture [25–27]. Recently, an LNA dT Blocker containing Locked Nucleic Acid (LNA) nucleotides  has been introduced. There is currently no standardized protocol available that can be used to evaluate and optimize blocking procedures with respect to optimizing the observed number of differentially expressed genes. In the present study we therefore compared the effect of poly(dA)40–60 and different doses of LNA dT blocker on the number of differentially expressed genes, and used the HCSSM to optimize identification of truly differentially expressed genes.
Results and discussion
The choice and dose of blocking reagent influences the number of differentially expressed genes estimated
In order to evaluate how different conditions designed to block poly(dA)-poly(dT) cross hybridization affect the ability to detect differentially expressed genes, we applied the HCSS-method to analysis of microarray experiments performed in the presence of either poly(dA)40–60 or varying amounts of LNA. Poly(dA)40–60 binds to the poly(dT) segment of the labelled target in the hybridization solution and thus competes with the poly(dA) segment in the microarray cDNA probes. LNA dT blocker contains Locked Nucleic Acid (LNA) nucleotides  at key positions within the (dT) synthetic strand, and is designed to block poly (dA) sequences present in the microarray cDNA probes and prevent them from hybridizing to poly(dT) segment of the labelled targets.
Background correction method has a stronger impact on the estimated number of differentially expressed genes than filtering
Validation of cDNA microarray data generated by HCSSM
Selected GO categories associated with genes differentially expressed in the cell lines AR42J versus NRK52E.
response to stimulus
response to stress
Cross platform microarray analysis
Since RT-PCR is still considered the "golden standard" for gene expression measurements, we further used SYBR green-based quantitative real-time PCR to validate the relative gene expression for a few of the 1478 additional genes identified with optimal conditions. Commonly, a 2 fold change is reported as the cut-off below which microarray and qRT-PCR data begin to loose correlation, although recent reports observed significant correlation where genes exhibited 1.4 fold change or higher [34, 35]. We therefore selected seven genes in the fold ratio range 4.8 to 1.8 (log2 ratio range 2.2 to 0.9 on the cDNA arrays). Results are shown in Additional files 3 and 4. Description of PCR primers and qRT-PCR protocol are shown in Additional file 5. Five of the selected genes (Ica1, c-fos, Btg2, Uhrf and Ube2b) were detected as differentially expressed on both microarray platforms. Two genes (Hoxa2b and Irfrd) were only identified on the the cDNA platform. Five genes with fold ratio > 2 were verified by qRT-PCR (see Additional files 3 and 4). Two genes (Hoxa2 and Ube2b) with fold ratio < 2 were not verified. Hox2b was excluded from the results due to technical problems (see Additional file 4). The gene Ube2b was expressed in the opposite direction relative to the cDNA microarray, but in the same direction as on the Illumina platform (higher in AR42J than in NRK52E on Illumina, lower on cDNA). Consistent with other reports [35, 36], the microarray ratios were compressed compared to qRT-PCR results (see Additional file 3). Overall, the qRT-PCR measurement corresponded 100 % (5/5) with the Illumina data and 71 % (5/7) with the cDNA microarray data. The latter confirmation rate is in accordance with cDNA microarray results of others (, and references therein).
Some of the discrepancy between cDNA microarry versus Illumina and qRT-PCR may be due to errors (reported by e.g. ) in the IMAGE collection used for probes in our cDNA microarrays. We thus consider the results from qRT-PCR and the Illumina platform more likely to be "true data" in the present study. In spite of the known errors on cDNA platforms, cross platform analysis confirmed 72 % of the results. Validation of a few selected genes by qRT-PCR supported this finding. Taken together, functional annotation analysis and the alternative methods for validation of the cDNA micoarry data indicated that the increase in the number of differentially expressed genes found by HCSSM was mainly real.
In the present study we have used the HCSSM in cDNA microarray control experiments to evaluate blocking conditions, filtration and background correction. We found that the choice and dose of poly(dT)-poly(dA) blocking agents during hybridization influenced the estimated number of differentially expressed genes. When HCSSM was used to evaluate steps in the computational analysis of microarray data we found that no background correction and weight based filtering gave the largest estimated numbers of differentially expressed genes. The background correction method, however, has a stronger impact on the numbers of differentially expressed genes found than filtering. The results show that HCSSM may be a useful and simple approach to optimize cDNA microarrays procedures without including external standards.
AR42J (rat pancreatic acinar cell derived, ATCC, Rockville, MD) were grown in Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/l glucose (Invitrogen, Carlsbad, CA), 15 % fetal calf serum (Euroclone Ltd, Devon, UK), 1 mM sodium pyruvate, 0.1 mg/ml L-glutamine (Invitrogen, Carlsbad, CA), 10 U/ml penicillin/streptomycin (Invitrogen, Carlsbad, CA), and 1 μ g/ml fungizone (Invitrogen Carlsbad, CA). NRK52E cells (rat kidney epithelial cells, ATCC, Rockville, MD) were grown in DMEM with 4.5 g/l glucose (Invitrogen, Carlsbad, CA) supplemented with 5 % FCS (Euroclone Ltd), 0.1 mg/ml L-glutamine (Invitrogen, Carlsbad, CA) and 10 U/ml penicillin/streptomycin (Invitrogen, Carlsbad, CA). All cells were grown at 37°C in a humidified atmosphere at 5 % CO2.
Treatment of cells and isolation of RNA
AR42J and NRK52E cells were cultured in 75 cm2 culture flasks for 72 h until confluence was reached. Total RNA was isolated using RNeasy® Midi kit (Qiagen, Germantown, MD) according to the manufacturer's instruction. RNA was quantified and assessed for purity by measurement of OD260 and OD280 using a UV fiberoptic spectrophotometer (Nanodrop Technologies, Rockland, DE) and was qualitatively assessed by measurement of relative 28S and 18S ribosomal band intensities using a Bioanalyzer and RNA LabChip capillary gel electrophoresis assay (Agilent Technologies, Palo Alto, CA). Aliquots were kept frozen at -80°C until further processing.
cDNA Microarray procedure
cDNA Microarrays were manufactured by the Norwegian Microarray Consortium  using 15000 cDNA rat probes from Research Genetics (IMAGE collection) printed in duplicates on Corning CMT Gaps II slides (Corning Inc. NY). The probes were dissolved in 50 % DMSO to ensure high printing quality. The microarray slides were UV cross linked at 300 mJ in order to fix the DNA to the glass slides.
Labelling was performed by reverse transcription of total RNA from the cell lines (3 μ g each) in the presence of primers containing the capture sequence for subsequent hybridization of Cy3- and Cy5-labelled dendrimers, using the Genisphere 3DNA Array 350 Expression Array Detection kit (Genisphere, Hatfield, PA) as described in the manufacturer's protocol. Different combinations of blocking reagents were added to the hybridization mixture: Mouse COT-1 DNA (Life Technologies) was added to all mixtures (0.1 μ g/μ g RNA) in order to block hybridization of repetitive elements. Different concentrations (0.1–4.0 μ l = 25–1000 ng) of LNA dT Blocker (Genisphere, Hatfield, PA) and 1 μ l = 1000 ng Poly(dA)40–60 (Stratagen Spotreporter; La Jolla, CA) were used as illustrated in Figure 2.
Hybridization was done in a humidified hybridization chamber (Corning Inc., NY) at 60°C for 14–15 h in a total hybridization volume of 60 μ l. Post hybridization washes were done for 15 min at 55°C with 2× SSC and 0.2 % SDS, for 10 min at room temperature with 2× SSC, and finally for 10 min at room temperature with 0.2× SSC. After the washing, the Cy3- and Cy5-labelled dendrimers were hybridized to the capture sequence at the reverse transcribed sample RNA at 60°C for 3 h as described in the manufacturer's protocol. After dendrimer hybridization, the slides were washed as described above and dried by centrifugation at 1500 rpm for 5 min.
Scanning and image analysis of cDNA microarrays
The slides were scanned at a resolution of 10 μ m by use of Packard Bioscience Scanarray Express HT scanner (Packard BioScience, Billerica, MA). A laser power of 100 % was used, and excitation of Cy3 and Cy5 was performed at a wavelength of 532 nm and 635 nm, respectively. Signals were detected by use of photomultiplier tubes (PMTs) with two channels. In order to maximizing the certainty of the weakest spots, the PMT voltage was adjusted to keep the background intensity between 200 and 400 (mean spot intensity) in each channel as suggested in . The GenePix 5.0 image analysis software (Axon Instruments, Inc., Union City, CA) was used for spot segmentation and intensity calculations. Spots and regions with high unspecific binding of dye or dust particles were manually flagged and excluded from the analysis.
Data analysis of cDNA microarrays
Modified T-scores and false discovery rate (FDR) values were calculated as described under Applications in the Background section for all combinations of filter, background corrections, and blocking treatments.
A progression of increasingly complex filter functions was evaluated.
Coarse filter: Spots flagged as bad or missing during the image analysis are removed.
Medium filter: Also removes spots with intensities below 200 and spots with more than 70 % saturation in both channels.
Fine filter: Also removes spots that are small, i.e. spots with diameters less than 20.
Uncertain filter: Removes spots flagged by GenePix 5.0 as "bad" or "missing" and spots with deltaM > 3.
The delta term is a small number added to stabilize spots with very small uncertainties.
Background correction methods
Three different background corrections strategies were applied:None were only foreground signal is used to calculate ratios, Edwards which is a subtraction method that ensures positive values  and dampened Edwards were a small number (e.g. 20) is added to the corrected signal to avoid extreme ratios in those cases where the background corrected intensity would be very low.
After filtering and background correction, all arrays were normalized using loess to remove intensity dependent variations in the ratios . Differentially expressed genes were found at the 5 % FDR level using the methodology described in the Background section.
Cross-platform microarray analysis
The Illumina Gene Expression system was used for cross-platform analysis of differentially expressed genes in AR42J cells versus NRK52E cells. RNA amplifications and hybridization were performed at the Finnish DNA Microarray Centre at Turku Centre of Biotechnology . Briefly, aliquots from the same RNA as in the cDNA microarray experiments were amplified with Ambion's Illumina® TotalPrep RNA Amplification kit (cat no AMIL1791) using 400 ng of total RNA as input material. The in vitro transcription (IVT) amplification that incorporated biotin-labelled nucleotides was performed overnight (14 h) at 37°C. After the amplifications the cRNA concentrations where checked with NanoDrop ND-1000 and cRNA quality was controlled by BioRad's Experion electrophoresis station.
A total of 750 ng of each biotin-labelled cRNA sample was hybridized to Illumina's Sentrix® RaRef-12-v1 Expression BeadChips at 58°C overnight (17 h) according to the manufacturer's instructions. The hybridized biotinylated cRNA was detected with 1 μ g/ml Cyanine3-streptavidine, (GE Healthcare Biosciences; cat no PA43001) and the Beadchips were scanned with Illumina BeadArray Reader (Factor = 1, PMT = 521, Filter = 100 %). Numerical results were extracted with Bead Studio v126.96.36.199 without any normalization or background subtraction.
The Illumina data were analysed using a modified T-test  with p-values adjusted for false discovery rate , and genes with adjusted p-values < 0.05 were taken as significant. Entrez Gene IDs were used as common identifiers for the microarray platforms. The genes were identified according to UniGen build #163 in GeneTools [29, 30].
Database submission of microarray data
The microarray data were prepared according to minimum information about a microarray experiment (MIAME) recommendations  and deposited in the Array Express . Detailed information about the microarray designs (platforms) and raw data files from the experiments are accessible in ArrayExpress by use of these accession numbers; A-MEXP-358 (cDNA platform), E-TABM-202 (cDNA experiment) and E-TABM-307 (Illumina experiment).
This work was supported by The Norwegian Research Council, The Norwegian Cancer Association, The Cancer Research Fund at St. Olav's Hospital, and the scientific collaboration between NTNU and St. Olav's University Hospital (Samarbeidsorganet). The cDNA microarray work was done with support from the national microarray technology platform (NMC), which is supported by the functional genomic program (FUGE). The Illumina work was performed at the Finnish DNA Microarray Centre at Turku Centre of Biotechnology .
We thank Bob Getts from Genisphere for helpful discussion on optimizing and evaluation of blocking conditions, Kristine Misund and Tonje S. Steigedal from NTNU for designing qRT-PCR primers and Anne Kristensen from NTNU for skilful technical assistance.
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