Volume 7 Supplement 2
Improvement in the Reproducibility and Accuracy of DNA Microarray Quantification by Optimizing Hybridization Conditions
© Han et al; licensee BioMed Central Ltd. 2006
Published: 26 September 2006
DNA microarrays, which have been increasingly used to monitor mRNA transcripts at a global level, can provide detailed insight into cellular processes involved in response to drugs and toxins. This is leading to new understandings of signaling networks that operate in the cell, and the molecular basis of diseases. Custom printed oligonucleotide arrays have proven to be an effective way to facilitate the applications of DNA microarray technology. A successful microarray experiment, however, involves many steps: well-designed oligonucleotide probes, printing, RNA extraction and labeling, hybridization, and imaging. Optimization is essential to generate reliable microarray data.
Hybridization and washing steps are crucial for a successful microarray experiment. By following the hybridization and washing conditions recommended by an oligonucleotide provider, it was found that the expression ratios were compressed greater than expected and data analysis revealed a high degree of non-specific binding. A series of experiments was conducted using rat mixed tissue RNA reference material (MTRRM) and other RNA samples to optimize the hybridization and washing conditions. The optimized hybridization and washing conditions greatly reduced the non-specific binding and improved the accuracy of spot intensity measurements.
The results from the optimized hybridization and washing conditions greatly improved the reproducibility and accuracy of expression ratios. These experiments also suggested the importance of probe designs using better bioinformatics approaches and the need for common reference RNA samples for platform performance evaluation in order to fulfill the potential of DNA microarray technology.
DNA microarray has become the major tool to study global gene expression profiles in recent years [2, 3]. Data from microarray experiments have been successfully used for establishing new pathways and identifying "signature" genes to differentiate cell types [4, 5]. Because of the increased use of microarrays to analyze the gene transcriptional response, it is crucial to ensure the reproducibility, reliability, and accuracy of microarray data.
DNA microarray is a very complex process involving many steps, such as probe design, array fabrication, RNA labeling, hybridization and washing, scanning, and data acquisition. Any missteps in the microarray process may lead to noise in the microarray experiment, which would adversely affect any conclusions drawn from the experiment. Various issues have been raised about the reliability and validity of microarray gene expression data [6–8]. For example, sub-optimally designed probes or incorrect probe annotations can lead to unreliable measurements in microarray experiments . At a more fundamental level, a lack of consistency within and between different microarray platforms when the same RNA samples were tested has also been reported [6–8, 10–12]. Such reports cast suspicion on microarray results and conclusions. Recent studies have shown, however, that carefully following established protocols, and using robust experimental designs and appropriate analytical methods can reduce the variability in microarray experiments and can result in much higher reproducibility and consistency [13–16]. In addition, there are many technical issues that must be controlled in the fabrication and use of spotted microarrays that can have a dramatic impact on the quality of microarray data . For example, intra-lab consistency can be improved by (1) the optimization of printing conditions such as relative humidity and buffer composition [18, 19], (2) the optimization of purification procedures for RNA amplification and labeling [20, 21], and (3) using consistent scanner power and voltage settings [22–24].
The fundamental basis of microarray technology is the specific binding (hybridization) of each probe to a labeled complementary target during the hybridization process . The specificity of each oligonucleotide probe is associated with its melting temperature (Tm) and the salt concentration in the hybridization buffer. Well-designed oligonucleotide sets should have a very narrow Tm range to ensure all the probes have very similar hybridization properties under the chosen hybridization condition.
In this paper, we used tissue and mixed tissue RNA samples to assess the effect of hybridization and washing conditions on the microarray expression ratios. The reproducibility and accuracy (specificity) of microarray data were greatly improved with the optimized hybridization and washing conditions. These experiments also suggest that improvements in probe design will improve the reliability of microarray measurements and the ability to extract meaningful information from microarray data.
Detection of non-specific binding under manufacturer-recommended hybridization condition
Average log2 ratios (Mix1/Mix2) of tissue selective genes of the mixed tissue RNA reference materials .
0.12 ± 0.21
0.06 ± 0.22
0.16 ± 0.21
-0.06 ± 0.40
0.43 ± 0.24
0.01 ± 0.10
0.30 ± 0.21
-0.87 ± 0.58
1.00 ± 0.28
0.24 ± 0.22
0.65 ± 0.25
-1.31 ± 0.23
Effect of washing condition stringency on DNA microarray expression ratios
To compare the effect of washing condition alone on the microarray expression ratios, rat liver RNA (Cy5, NCTR) and Universal Rat Reference RNA (Cy3, Stratagene) were hybridized to the 4 k rat oligonucleotide arrays (Clontech) using GlassHyb™ buffer at 50°C for 16–18 hours, but washed differently: one with washing condition 1 and the other with the more stringent washing condition 2 (see Materials and Methods). The results showed that the signal-to-background ratios of lambda spots dropped slightly with the more stringent washing condition (Figures 3A and 3B). However, the lambda spots still gave higher signal-to-background ratios than the rat-specific oligonucleotide probes. Because of the relatively small improvement in signal-to-background ratios as a result of making the washing conditions more stringent, we focused on adjusting the hybridization conditions.
Effect of hybridization condition on DNA microarray expression ratios
Since the Clontech oligonucleotide probe sequences and the composition of their hybridization buffer GlassHyb™ are proprietary and unknown to us, rat liver RNA (Cy5, NCTR) and Universal Rat Reference RNA (Cy3, Stratagene) were hybridized to the rat 4 k arrays (Clontech) using a hybridization buffer composed of 5× SSC, 0.1% SDS and 32% formamide. The hybridization buffer was defined using the equation Tm = 81.5 + 16.6 (log10 M) + 0.41 (% GC) - 0.61 (% form) - 500/L, where Tm is the melting temperature, M is the molarity of Na+, L is the length of base pairs, and % form is the percentage of formamide . It was assumed that the 80-mer oligonucleotide probes from Clontech contained an equal number of A, T, C, and G bases. The hybridization temperature was targeted about 20°C below the Tm. The hybridization temperature was set to 50°C so adjustments were made in the Na+ and formamide concentration to meet these criteria. The slides were washed with washing condition 1. The results (Figure 3C) showed that the stringent hybridization conditions dramatically reduced the signal-to-background ratios of the lambda sequences while maintaining high signal-to-background ratios for the rat sequences. Thus, the hybridization condition appeared to play a greater role than the washing condition in reducing non-specific binding between the labeled targets and the oligonucleotide probes on the microarray.
Discrepancies between expression ratios from microarray and theoretical input ratios, and discrepancies between microarray platforms
Comparison of transcript expression in MTRRMs (Mix1/Mix2) by qRT-PCR and microarray.
To assess the performance of our in-house printed oligonucleotide microarrays, mixed tissue RNA reference materials (MTRRM) were labeled and hybridized with rat 4 k arrays under the oligonucleotide manufacturer's recommended condition. The compressed expression ratios for MTRRMs showed there was severe non-specific binding under the recommended hybridization condition. A series of experiments was designed to optimize the hybridization and washing conditions for the in-house printed arrays. With these optimized hybridization and washing conditions, non-specific binding was greatly reduced, and microarray data reproducibility and accuracy were highly improved. Without reference RNA samples, such as the MTRRM, it is very difficult to evaluate accuracy of microarray data. We have shown that very high reproducibility can be obtained under non-optimal hybridization conditions but that the accuracy was very low. The low stringency of the hybridization conditions would cause a failure to identify true changes in gene expression because of the highly compressed nature of the signals due to cross-hybridization. Only through use of calibrated RNA references can the accuracy be judged. The strategy employed by Thompson et al.  to generate calibrated rat RNA reference materials can be readily adapted to the production of reference RNA for any desired species, in particular human and mouse. Although discrepancies remained between the ratios measured in this study and the theoretical ratios for the tissue RNA components in the MTRRM, it should be noted that the tissue selectivity of probes recommended for use with the MTRRM was empirically determined for 3 commercial platforms (Agilent, Affymetrix, and CodeLink ), but not for the oligonucleotide set used here.
A DNA microarray experiment is a complex, multi-step process. The probe design, hybridization condition, and scanner settings are major factors determining the accuracy and reproducibility of the final specific signal quantification. Signals measured from individual probes on a microarray are a summation of the non-specific hybridization (mismatched cross-hybridization; imperfect matches between probes and targets) and specific hybridization (perfect matches between probes and targets). If the non-specific hybridization signal is relatively large and constant between samples, the ratio measurement between two samples will be compressed as shown in Figure 1. Thus, it is crucial for accurate gene expression measurements to reduce non-specific hybridization as we have described. To avoid non-specific binding (cross-hybridization), probe design has been one of the major focuses for the past few years [27–35]. Commercial microarray manufacturers and oligonucleotide providers have been updating and redesigning their oligonucleotide sets to improve the reliability and quality of the signals. The lack of reference RNA materials, however, has limited the examination of the accuracy of the probes under the conditions recommended by manufacturers.
Aside from probe design, the hybridization stringency (salt concentration, temperature, and pH) plays a critical role to ensure the specific binding of targets to their complementary probes . At a certain hybridization condition, these bindings are not only affected by the Tm's of each probe, but also by the concentration and the secondary structure of the target. This might explain the ratio discrepancies of some of the probes with similar Tm's in our results (Figure 9). The increasing number of probes printed on whole genome microarrays also increases the chance of cross-hybridization (non-specific binding). A recently published study that used a systematic multivariate approach to correct cross-hybridization signals in expression experiments  points out the limitations of current understanding of hybridization on solid supports and the consequent difficulty in designing optimal probes.
Recently the discrepancies between various microarray platforms have received great attention in the microarray community. Many studies have been done to address the comparability of different microarray platforms and contradictory conclusions have been reached, with some studies reporting good concordance [13–16] and others a lack of agreement [6–8, 10–12]. As in the present study, well-characterized reference RNAs may be valuable in resolving this debate. The FDA-led MicroArray Quality Control (MAQC; http://www.fda.gov/nctr/science/centers/toxicoinformatics/maqc/) project is developing such materials and using them to compare the reproducibility of the same microarray platform across sites as well as the comparability between different platforms. The outcome promises to provide a better picture in terms of performances and comparability among different microarray platforms. Meanwhile, development of sets of well-calibrated reference RNA samples, similar to the MTRRMs with expanded gene coverage, would also be useful for microarray performance evaluation. All of these efforts would greatly help to standardize the microarray process and maximize the potential of microarray technology.
The results from this study demonstrated the importance of hybridization optimization to generate highly reproducible and accurate microarray data. The use of MTRRMs and other dissimilar RNA types were shown to be effective tools for the optimization process.
Materials and methods
Microarray slides and sources of RNA samples
Rat 4 k (4000 oligonucleotide probes; Clontech, Palo Alto, CA), rat 10 k (10,000 rat oligonucleotide probes; MWG-Biotech, High Point, NC), and mouse 20 k (20,000 mouse oligonucleotide probes; MWG-Biotech) oligonucleotide sets were dissolved in MWG Spotting Buffer A (MWG-Biotech, High Point, NC) and printed on poly-L-lysine-coated slides (Erie Scientific, Portsmouth, NH) using an OmniGrid™ Microarrayer (GeneMachines, San Carlos, CA) at the National Center for Toxicological Research (NCTR). Printed slides were baked at 80°C for 1 hour and UV cross-linked with 300 mJoules (UV Stratalinker 2400, Stratagene, La Jolla, CA). Following this, the slides were treated with a blocking solution of 3× SSC, 0.1% SDS and 1% BSA using gentle agitation for 5 minutes at 50°C and washed with MilliQ water four times for 5 minutes each at room temperature. The slides were then placed in MilliQ water heated to boiling for 2 minutes, followed by a 1 minute wash in ethanol at room temperature. The slides were spun dry in a microarray high speed centrifuge (TeleChem International, Sunnyvale, CA).
RNA samples used in these experiments were from several different sources; rat liver RNA was extracted at NCTR. Rat mixed tissue RNA reference materials (MTRRM ) were provided by the U.S. FDA, Center for Drug Evaluation and Research. Mix1 and Mix2 contain different proportions of four tissue total RNAs. Mix1 contains total RNA from brain (40%), liver (30%), kidney (20%) and testes (10%); Mix2 contains total RNA from brain (20%), liver (20%), kidney (20%) and testes (40%). The theoretical ratios of the tissue selective genes (genes were predominately expressed in only one of the tissues in the mixture) for kidney, liver, brain, and testis between the two mixtures (mix1/mix2) were 1, 1.5, 2, and 0.25, respectively. Universal rat reference RNA (Stratagene, La Jolla, CA), rat brain total RNA and rat liver total RNA (Ambion, Austin, TX) were also used for performance evaluation.
RNA Isolation and cDNA Labeling
Total RNA was extracted from adult rat and mouse liver tissue using Qiagen RNeasy kits (Qiagen, Valencia, CA) with on-column DNase digestion. The concentrations and A260/A280 ratios of the total RNA samples were measured by NanoDrop ND-1000 spectrophotometry (NanoDrop Technologies, Wilmington, DE). The quality of the total RNA was further evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). RNA samples with RINs (RNA Integrity Number) above 8.0 were used for microarray analysis. All RNA samples were aliquoted at 10 μg per tube and frozen at -80°C until used for microarray analysis.
Target cDNAs were prepared by aminoallyl labeling followed by coupling of fluorescent dyes. Briefly, 10 μg of total RNA and 6 μg of random hexamer primers (Invitrogen, Carlsbad, CA) were mixed together to a final volume of 16.5 μl, incubated at 70°C for 10 minutes and snap frozen on dry-ice and ethanol for 1 minute. The RNA was reverse transcribed in a 30 μl reaction containing 0.5 mM dATP, dCTP, dGTP, 0.3 mM dTTP (Invitrogen), 0.2 mM aminoallyl-dUTP (aa-dUTP, Sigma, St. Louis, MO), 40 U RNase Inhibtor (Invitrogen), 400 U superscript II (Invitrogen), 10 mM DTT and 1X first strand buffer (50 mM Tris-HCl, 75 mM KCl, and 5 mM MgCl2, pH 8.3). This mixture was incubated at 42°C for 2 hours to generate aminoallyl-labeled cDNA. The RNA templates were hydrolyzed at 65°C for 15 minutes using 10 μl of 1 N NaOH and 10 μl of 0.5 mM EDTA. The reaction was neutralized by adding 10 μl of 1 N HCl. Unincorporated aa-dUTP was removed by QIAquick PCR purification kit (Qiagen) and the aminoallyl-cDNAs were then dried in a SpeedVac System SPD 1010 (Themo Savant, Holbrook, NY) at 45°C. Aminoallyl-cDNA pellets were resuspended in 4.5 μl of 0.1 M sodium carbonate buffer (pH 9.0) and coupled with 4.5 μl of Cy3 or Cy5 monoreactive dyes (approximately 24 nmoles of dyes) (Amersham Pharmacia, Piscataway, NJ) for 1 hour at room temperature in the dark. Monoreactive Cy3 and Cy5 dyes supplied in each vial were resuspended in 73 μl of DMSO and aliquoted into 16 tubes of 4.5 μl each and one aliquot was used for each labeling reaction. Uncoupled dyes were removed by QIAquick PCR purification kit (Qiagen). cDNA yields and dye incorporation efficiencies were determined using the NanoDrop ND-1000 spectrophotometer.
Hybridization and washing conditions
Cy3 and Cy5 labeled cDNAs were mixed together and concentrated to less than 5 μl using a SpeedVac SPD 1010 at room temperature. The samples were then mixed with 60 μl of pre-warmed hybridization buffer, either GlassHyb™ (Clontech) or 5× SSC (Ambion), 0.1% SDS (Sigma) with various formamide (Invitrogen) concentrations at 50°C. The labeled cDNAs were denatured at 95°C for 3 minutes and then placed in a water bath at 50°C until hybridization. The hybridizations were performed in ArrayIt hybridization cassettes (TeleChem International) in a water bath at 50°C for 16–18 hours.
Slides were washed under various stringency conditions. Washing condition 1: first wash: 2 × SSC containing 1% Tween-20 (Sigma, St. Louis, MO) at room temperature for 10 minutes on a shaker with gentle agitation; second wash: 1 × SSC containing 0.1% Tween-20 at room temperature for 5–10 minutes on a shaker with gentle agitation; third wash: 0.1 × SSC at room temperature for 5–10 minutes on a shaker with gentle agitation.
Washing condition 2: first wash: 0.3 × SSC containing 1% Tween-20 at 50°C for 10 minutes on a shaker with gentle agitation; second wash: 1 × SSC containing 0.1% Tween-20 at 50°C for 5–10 minutes on a shaker with gentle agitation; third wash: 0.1 × SSC for 5–10 minutes on a shaker with gentle agitation.
Washing condition 3: first wash: 2 × SSC containing 1% SDS at 30°C for 5 minutes on a shaker with gentle agitation; second wash: 1 × SSC at 30°C for 5 minutes on a shaker with gentle agitation; third wash: 0.5 × SSC at 30°C for 5 minutes on a shaker with gentle agitation. The hybridized slides were dried immediately by centrifugation after the final wash step.
Scanning, Feature Extraction and Data Analysis
The hybridized slides were scanned with a GenePix 4000B scanner (Axon Instruments, Union City, CA) at 10 μm resolution using appropriate photomultiplier tube gains to obtain the highest intensity with <0.1% saturated pixels. The resulting images were analyzed by measuring the fluorescence of all features on the slides using the GenePix Pro 6.0 image analysis software (Axon Instruments). The median fluorescence intensity of all the pixels within one feature was taken as the intensity value for that feature. All the raw data were imported into ArrayTrack  and were normalized using Total Intensity Normalization or LOWESS Normalization with background subtraction. Student's T-test was used to compute p values.
We thank the US FDA Office of Science and Health Coordination for their support. The views presented in this article do not necessarily reflect those of the Food and Drug Administration.
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