Signal stability of Cy3 and Cy5 on antibody microarrays
© Gu et al; licensee BioMed Central Ltd. 2006
Received: 22 June 2006
Accepted: 11 October 2006
Published: 11 October 2006
The antibody microarray technique is a newly emerging proteomics tool for differential protein expression analyses that uses fluorescent dyes Cy 3 and Cy 5. Environmental factors, such as light exposure, can affect the signal intensity of fluorescent dyes on microarray slides thus, it is logical to scan microarray slides immediately after the final wash and drying processes. However, no research data are available concerning time-dependent changes of fluorescent signals on antibody microarray slides to this date. In the present study, microarray slides were preserved at -20°C after regular microarray experiments and were rescanned at day 10, 20 and 30 to evaluate change in signal intensity.
Fluorescent intensities of microarray spots were detected using a confocal laser scanner after the experiment at day 0, and re-examined at day 10, 20 and 30, respectively. Fluorescent intensities of rescanned microarray spots did not show significant changes when compared with those scanned immediately after standard microarray experiments.
Microarray slides can be preserved and rescanned multiple times using a confocal laser scanner over a period of days or weeks.
Antibody microarray analyses of protein expression levels represent a new trend of cutting-edge proteomics research [1–6], and have been increasingly utilized in studies of normal and pathological conditions [7–22]. The technique has several distinct advantages. First, compared to the cDNA microarray analysis, antibody microarrays detect differential gene expression at the protein level. Although cDNA microarray analyses have generated a large database concerning gene expression patterns, most of these studies have focused on gene expression at the mRNA level only, with the assumption that the relative mRNA levels represent the relative levels of proteins. Since there is often a poor correlation between mRNA levels and protein levels [23–28], this assumption may not be true for many of the genes. Second, the antibody microarray technique is more sensitive compared to gel electrophoreses. With microarray technology, protein levels can be detected in the low pg/ml range. This allows the measurement of the expression of both small and large molecular weight proteins simultaneously, regardless of their isoelectric points. In addition, some protein extraction buffers used for antibody microarray experiments contain non-denaturing detergents in order to keep the proteins in their native state. While gel electrophoreses usually separate denatured proteins for expression analyses, antibody microarrays can measure relative abundance of naïve undenatured proteins.
The vast majority of antibody microarray experiments conducted so far used the fluorescent dyes Cy3 and Cy5 for protein labelling. In general, fluorescent dyes are sensitive to light exposure as well as other environmental factors such as water, high temperature, alkali, and alcohol. Recent evidence indicates that even ozone levels in the laboratory atmosphere could affect fluorescent dyes on microarrays . In order to avoid or to minimize the effects of these risk factors, it is appropriate to scan microarray slides instantly upon finishing the final wash and drying processes without unnecessary delays. However, no data are currently available concerning time-dependent changes of fluorescent signals on antibody microarray slides. Furthermore, the published research on repeated scans of antibody or protein microarrays is scant. Although repeated scans of cDNA microarrays have been conducted [30–35], it is not known whether amine-coupled fluorescent dyes on antibody/protein microarrays behave in the same way as those nucleotide-coupled fluorescent dyes on cDNA microarrays. In addition, previous studies conducted multiple scans of cDNA microarrays sequentially, usually within minutes. Therefore, it is also unknown whether prolonged intervals between scans in the range of days or weeks could modify the signal intensity of fluorescent dyes on antibody or protein microarrays. Furthermore, previous rescan studies were conducted using different laser power levels or photomultiplier tube (PMT) gains rather than using a consistent setting of the laser power and PMT. It is expected that different settings of the laser power and PMT generated different signal intensities. Whether the same setting of the laser power and PMT can generate consistent intensity outcome over time has not been demonstrated. Using a confocal laser scanner to minimize photo-bleaching effects, we scanned microarrays slides at day 0, 10, 20 and 30, respectively. After regular antibody microarray experiments, the microarray slides were stored in a laboratory freezer at -20°C. Fluorescent intensities of microarray spots at these time points were then quantified and compared. Our results indicate no significant changes in intensities of both Cy3 and Cy5 signals over the examined period.
Results and discussion
Clontech™ antibody microarrays were used in this study, which had 1024 microarray spots on each slide. Among them, 6 spots were printed with fluorescence-labeled albumin and served as positive controls, whereas 4 spots were printed with non-labeled albumin and served as negative controls. For all microarray slides, the negative control spots had similar intensities as those of background signals. These 10 control spots were not included in the final microarray spot analyses. The remaining 1014 spots on each microarray slide were followed up throughout the entire length of the study. In order to keep the same experimental condition, the following steps were taken: when a storage vial containing the microarray slide was taken out of the freezer, it was kept at room temperature for 30 minutes; and to keep the scan condition consistent, the same strength of the laser power (100%) and PMT (65%) were used for all scans of the microarray slides. No spot on the microarray slides was saturated using these settings of the laser power and PMT, so that unchanged intensities of microarray spots due to saturation could be excluded in the present study.
For each microarray spot, the signal intensity was measured 4 times, at day 0 (I0), day 10 (I10), day 20 (I20), and day 30 (I30), respectively. Because I0 of each microarray spot had a different value, we set I0 as the reference, and calculated the ratio of I10/I0, I20/I0, and I30/I0, respectively, for each of the 1014 spots on the microarray slide. If the fluorescent signals on a microarray slide decrease over time, the ratio value of I10/I0, I20/I0, and I30/I0 should become smaller and smaller. Fourteen slides were examined for Cy3 and for Cy5, respectively.
Summary of ratio comparisons of Cy3 spots.
1.037 ± 0.004
1.086 ± 0.005
1.157 ± 0.006
1.008 ± 0.017
1.047 ± 0.004
1.087 ± 0.004
0.986 ± 0.003
1.025 ± 0.003
1.006 ± 0.002
1.021 ± 0.002
1.093 ± 0.002
0.988 ± 0.002
0.973 ± 0.007
1.006 ± 0.008
1.071 ± 0.009
1.044 ± 0.006
1.121 ± 0.007
1.104 ± 0.007
0.991 ± 0.008
0.990 ± 0.003
0.990 ± 0.003
0.982 ± 0.003
0.929 ± 0.003
0.948 ± 0.003
1.039 ± 0.004
1.051 ± 0.004
1.060 ± 0.004
1.055 ± 0.004
1.064 ± 0.006
1.001 ± 0.004
1.084 ± 0.009
1.142 ± 0.004
1.075 ± 0.004
1.066 ± 0.003
1.117 ± 0.012
1.026 ± 0.003
1.135 ± 0.007
1.138 ± 0.006
1.083 ± 0.021
0.985 ± 0.004
1.101 ± 0.003
1.114 ± 0.014
1.029 ± 0.013
1.065 ± 0.018
1.051 ± 0.017
Summary of ratio comparisons of Cy5 spots.
1.115 ± 0.013
1.102 ± 0.013
1.054 ± 0.013
1.139 ± 0.005
1.162 ± 0.007
1.141 ± 0.006
1.103 ± 0.006
1.185 ± 0.006
0.999 ± 0.072
1.150 ± 0.022
1.153 ± 0.020
1.053 ± 0.012
1.054 ± 0.007
1.133 ± 0.008
1.180 ± 0.009
1.170 ± 0.007
1.220 ± 0.008
1.191 ± 0.008
1.120 ± 0.006
1.093 ± 0.006
1.070 ± 0.012
0.999 ± 0.011
0.970 ± 0.003
1.010 ± 0.082
1.030 ± 0.004
0.978 ± 0.004
0.943 ± 0.004
1.055 ± 0.006
1.005 ± 0.007
1.054 ± 0.009
0.959 ± 0.005
0.957 ± 0.037
0.868 ± 0.003
0.902 ± 0.002
0.844 ± 0.002
0.814 ± 0.003
1.080 ± 0.006
0.996 ± 0.006
1.025 ± 0.007
0.932 ± 0.002
0.797 ± 0.003
0.828 ± 0.003
1.058 ± 0.024
1.042 ± 0.037
1.016 ± 0.034
Microarray spot signal intensities can span a broad range from 0 to 65536 (= 216). To examine whether microarray spots with different intensities behave differently over time, we divided the microarray spots into three groups according to their intensities: 1) lower range when I0 was lower than 20000, 2) middle range when I0 was between 20000 and 40000, and 3) higher range when I0 was higher than 40000, and calculated spot intensities in each group of the Cy3-labeled (N = 14 slides) and in each group of the Cy5-labeled (N = 14 slides) at day 0, 10, 20, and 30, respectively. Quantitative analyses did not show statistically significant changes in intensities over the examined time frame regardless of their intensities. (p > 0.05, ANOVA).
These results suggest that Cy3 and Cy5 signals on antibody microarrays are stable when the microarray slides are stored in an airtight slide vial at -20°C. These results are crucial to guide contemporary proteomics research involving microarrays. For instance, occasionally microarray slides could not be scanned soon after the experiment due to time restrains. If this is inevitable, microarray slides could be stored and scanned at a later time. Oftentimes, it is determined that signals of some proteins on microarray slides were too strong (e.g. saturated) after data analysis has been done. In such cases, a rescan of microarray slides is usually desirable with a reduced setting of the laser power and/or PMT, so that signal intensities of these proteins become non-saturated and suitable for data analyses.
The signal intensities of microarray spots did not show a significant decrease after repeated scans, suggesting that the employed settings of the laser power and PMT were adequate for multiple scans. We used a confocal microarray scanner in our study, which, due to the confocal nature of the laser beam, kept the bleaching effect of the laser light minimal. However, whether a non-confocal microarray scanner could achieve similar results as a confocal scanner remains to be determined. Also, we tested only Cy3 and Cy5 dyes in the present study. It is possible that other fluorescent dyes may show similar results as those of Cy3 and Cy5.
In theory, the fluorescent signal on microarray slides should either remain the same or decrease in intensity but is not expected to increase over time. However, we had a few slides that displayed higher intensities at a later scanning time point when compared to the intensities acquired immediately after the drying procedure. Two possibilities may be accountable for this phenomenon. Such event could be attributed to the instability of the laser power and/or PMT of the microarray scanner. Another potential source of error may be due to incomplete drying of a microarray slide after the final centrifugation. Any remaining fluid in the slide holder may evaporate and keep the microarray slide humid. The moisture on microarray spots is reduced over time, which leads to a stronger fluorescent signal.
In the present study we employed antibody microarray slides from Clontech Laboratories Inc. (Mountain View, California) for the following reasons: The Clontech™ antibody microarrays detect a wide variety of cytosolic, membrane-bound, and nuclear proteins. Over 500 proteins can be examined in a single experiment. As part of the antibody microarray development, all antibodies have been extensively tested to verify their specificity. Each is raised against a known protein. Antibodies that display a poor specific signal or a high level of cross-reactivity were excluded from the antibody microarrays. In addition, all antibodies were checked for the linearity of signal that can be obtained, a necessary step to ensure accurate quantification. Antibodies that show non-linear binding kinetics were also excluded from the antibody microarrays. Each antibody is double printed side-by-side on the microarray slide to provide an additional internal control. The antibody microarrays are produced on standard 75 × 25 × 1 mm glass slides, an open platform that is compatible with commercially available scanners commonly used for DNA microarrays. Thus, the Clontech™ antibody microarrays represent the most comprehensive antibody microarrays to this date. Since the coupling of cyanine dyes (N-hydroxysuccinimide-esters) to proteins is universal through amines, our results should be applicable to other types of antibody or protein microarrays as well.
A recent study showed that ozone levels in the laboratory atmosphere could adversely affect intensities of fluorescent dyes such as Cy5 and Alexa 647 on DNA microarrays . The fact that fluorescent intensities did not show significant decrease at day 10, 20, and 30 when compared to those at day 0 suggests that the ozone level in our laboratory is not a critical component in our antibody microarray experiments.
Based on our experience we summarize the key issues to maintain the stability of fluorescent signals on microarray slides as follows: 1) to dry the microarray slides thoroughly, 2) to keep them under a dry condition, 3) to store them in a freezer (e.g. -20°C), and 4) to minimize light exposure.
Microarray spot signals are more stable than previously thought if microarray slides are stored at -20°C in an airtight slide vial. Microarray slides can be preserved and rescanned multiple times using a confocal laser scanner over a period of days or weeks.
Proteins extracted from visual cortex of young and adult mice were used in this study. The use of animals and the experimental procedures involving animals were approved by the Animal Care and Use Committee of Wake Forest University Health Sciences (Protocol number A03-045). All animals were euthanized with an overdose of pentobarbital (150 mg/kg body-weight). A conventional two-color dye-swap protocol was used for antibody microarray experiments [11, 17, 18, 20, 21]. In brief, proteins were extracted using a protein extraction buffer (Clontech, Mountain View, California), which contained non-denaturing detergents. Tissue samples were homogenized with alumina (0.5 g/100 mg tisue) and extraction buffer (2 ml/100 mg tissue). The suspension was centrifuged at 10,000 × g for 30 minutes at 4°C. The supernatant was collected and its protein concentration was measured using a Protein Assay Kit (Pierce, Rockford, Illinois). The protein concentration was diluted to 1.1 mg/ml by adding an appropriate volume of the extraction buffer. The monoamine reactive dyes Cy3 and Cy5 (Amersham, Piscataway, New Jersey) were dissolved in 110 μl extraction buffers, respectively. 50 μl dye solutions and 450 μl protein solutions were mixed to generate 4 samples: Adult-Cy3, Young-Cy3, Adult-Cy5, and Young-Cy5. After 90 minutes of incubation on ice, the labeling process was stopped by adding 4 μl of 1 M ethanolamine. After protein labeling, unbound dyes were removed by gel filtration using disposable PD-10 desalting columns (Amersham). Each column was equilibrated with 3 × 5 ml desalting buffer (Clontech) before adding a protein sample, which was eluted by applying 2 ml desalting buffer. The protein concentration in each sample was determined using the Pierce Protein Assay Kit.
Two antibody microarray incubation solutions were made with the following compositions: (1) 5 ml incubation buffer (Clontech), 25 μg of the Adult-Cy3 protein sample, and 25 μg of the Young-Cy5 protein sample, and (2) 5 ml incubation buffer, 25 μg of the Adult-Cy5 protein sample, and 25 μg of the Young-Cy3 protein sample. After 30 minutes incubation at room temperature with gentle rocking, the antibody microarray slides (Clontech) were washed with seven different wash buffers (Clontech) at 5 minutes each, dried by centrifugation (1,000 × g) in a swing bucket rotor at room temperature for 25 minutes, and scanned using a confocal microarray scanner (ScanArray Gx) with a pixel resolution of 5 μm. The excitation wavelength and the emission filter wavelength for Cy3 and Cy5 were preset by the scanner's manufacturer (Perkin-Elmer, Shelton, Connecticut) at 543 nm/570 nm (excitation/emission) and 633 nm/670 nm (excitation/emission), respectively. After each scan, the microarray slide was put in a dry and air-tight plastic vial, and stored in a -20°C freezer until the next scan. Fluorescent signals of microarray spots were quantified using ScanArray Express (Perkin-Elmer). The mean intensity of the fluorescent signal within each microarray spot as well as the mean background intensity immediately surrounding the microarray spot was determined. The intensity of a microarray spot is defined as the value of the spot mean intensity minus the background mean intensity. Analysis of variance (ANOVA) was applied to test statistic significance of signal intensities at different time points, using signal intensities of the microarray spots as dependent variables and the microarray spots (1014) and time points (4) as independent variables.
This work was supported by a grant from NIH (EY14892). We thank Veena Nagendrappa for writing Excel macros which were utilized in our data analyses.
- Haab BB: Methods and applications of antibody microarrays in cancer research. Proteomics 2003, 3: 2116–2122. 10.1002/pmic.200300595View ArticlePubMedGoogle Scholar
- Barry R, Soloviev M: Quantitative protein profiling using antibody arrays. Proteomics 2004, 4: 3717–3726. 10.1002/pmic.200300877View ArticlePubMedGoogle Scholar
- Perlee L, Christiansen J, Dondero R, Grimwade B, Lejnine S, Mullenix M, Shao W, Sorette M, Tchernev V, Patel D, Kingsmore S: Development and standardization of multiplexed antibody microarrays for use in quantitative proteomics. Proteome Sci 2004, 2: 9. 10.1186/1477-5956-2-9PubMed CentralView ArticlePubMedGoogle Scholar
- Wingren C, Borrebaeck CA: High-throughput proteomics using antibody microarrays. Expert Rev Proteomics 2004, 1: 355–364. 10.1586/147894184.108.40.2065View ArticlePubMedGoogle Scholar
- Angenendt P: Progress in protein and antibody microarray technology. Drug Discov Today 2005, 10: 503–511. 10.1016/S1359-6446(05)03392-1View ArticlePubMedGoogle Scholar
- Eckel-Passow JE, Hoering A, Therneau TM, Ghobrial I: Experimental design and analysis of antibody microarrays: applying methods from cDNA arrays. Cancer Res 2005, 65: 2985–2989.PubMedGoogle Scholar
- Belov L, de la Vega O, dos Remedios CG, Mulligan SP, Christopherson RI: Immunophenotyping of leukemias using a cluster of differentiation antibody microarray. Cancer Res 2001, 61: 4483–4489.PubMedGoogle Scholar
- Belov L, Huang P, Barber N, Mulligan SP, Christopherson RI: Identification of repertoires of surface antigens on leukemias using an antibody microarray. Proteomics 2003, 3: 2147–2154. 10.1002/pmic.200300599View ArticlePubMedGoogle Scholar
- Knezevic V, Leethanakul C, Bichsel VE, Worth JM, Prabhu VV, Gutkind JS, Liotta LA, Munson PJ, Petricoin EF 3rd, Krizman DB: Proteomic profiling of the cancer microenvironment by antibody arrays. Proteomics 2001, 1: 1271–1278. Publisher Full Text 10.1002/1615-9861(200110)1:10<1271::AID-PROT1271>3.0.CO;2-6View ArticlePubMedGoogle Scholar
- Lin Y, Huang R, Santanam N, Liu YG, Parthasarathy S, Huang RP: Profiling of human cytokines in healthy individuals with vitamin E supplementation by antibody array. Cancer Lett 2002, 187: 17–24. 10.1016/S0304-3835(02)00346-4View ArticlePubMedGoogle Scholar
- Anderson K, Potter A, Baban D, Davies KE: Protein expression changes in spinal muscular atrophy revealed with a novel antibody array technology. Brain 2003, 126: 2052–2064. 10.1093/brain/awg208View ArticlePubMedGoogle Scholar
- Miller JC, Zhou H, Kwekel J, Cavallo R, Burke J, Butler EB, The BS, Haab BB: Antibody microarray profiling of human prostate cancer sera: antibody screening and identification of potential biomarkers. Proteomics 2003, 3: 56–63. 10.1002/pmic.200390009View ArticlePubMedGoogle Scholar
- De Ceuninck F, Dassencourt L, Anract P: The inflammatory side of human chondrocytes unveiled by antibody microarrays. Biochem Biophys Res Commun 2004, 323: 960–969. 10.1016/j.bbrc.2004.08.184View ArticlePubMedGoogle Scholar
- Hudelist G, Pacher-Zavisin M, Singer CF, Holper T, Kubista E, Schreiber M, Manavi M, Bilban M, Czerwenka K: Use of high-throughput protein array for profiling of differentially expressed proteins in normal and malignant breast tissue. Breast Cancer Res Treat 2004, 86: 281–291. 10.1023/B:BREA.0000036901.16346.83View ArticlePubMedGoogle Scholar
- Turtinen LW, Prall DN, Bremer LA, Nauss RE, Hartsel SC: Antibody array-generated profiles of cytokine release from THP-1 leukemic monocytes exposed to different amphotericin B formulations. Antimicrob Agents Chemother 2004, 48: 396–403. 10.1128/AAC.48.2.396-403.2004PubMed CentralView ArticlePubMedGoogle Scholar
- Abdo M, Irving B, Hudson P, Zola H: Development of a cluster of differentiation antibody-based protein microarray. J Immunol Methods 2005, 305: 3–9. 10.1016/j.jim.2005.07.008View ArticlePubMedGoogle Scholar
- Andersson O, Kozlowski M, Garachtchenko T, Nikoloff C, Lew N, Litman DJ, Chaga G: Determination of relative protein abundance by internally normalized ratio algorithm with antibody arrays. J Proteome Res 2005, 4: 758–767. 10.1021/pr049776fView ArticlePubMedGoogle Scholar
- Bartling B, Hofmann HS, Boettger T, Hansen G, Burdach S, Silber RE, Simm A: Comparative application of antibody and gene array for expression profiling in human squamous cell lung carcinoma. Lung Cancer 2005, 49: 145–154. 10.1016/j.lungcan.2005.02.006View ArticlePubMedGoogle Scholar
- Gao WM, Kuick R, Orchekowski RP, Misek DE, Qiu J, Greenberg AK, Rom WN, Brenner DE, Omenn GS, Haab BB, Hanash SM: Distinctive serum protein profiles involving abundant proteins in lung cancer patients based upon antibody microarray analysis. BMC Cancer 2005, 5: 110. 10.1186/1471-2407-5-110PubMed CentralView ArticlePubMedGoogle Scholar
- Mathur P, Kaga S, Zhan L, Das DK, Maulik N: Potential candidates for ischemic preconditioning-associated vascular growth pathways revealed by antibody array. Am J Physiol Heart Circ Physiol 2005, 288: H3006-H3010. 10.1152/ajpheart.01203.2004View ArticlePubMedGoogle Scholar
- Mathur P, Kaga S, Zhan L, Das DK, Maulik N: Antibody-array technique reveals overexpression of important DNA-repair proteins during cardiac ischemic preconditioning. J Mol Cell Cardiol 2005, 38: 99–102. 10.1016/j.yjmcc.2004.11.032View ArticlePubMedGoogle Scholar
- Watanabe M, Guo W, Zou S, Sugiyo S, Dubner R, Ren K: Antibody array analysis of peripheral and blood cytokine levels in rats after masseter inflammation. Neurosci Lett 2005, 382: 128–133. 10.1016/j.neulet.2005.03.002View ArticlePubMedGoogle Scholar
- Anderson L, Seilhamer J: A comparison of selected mRNA and protein abundances in human liver. Electrophoresis 1997, 18: 533–537. 10.1002/elps.1150180333View ArticlePubMedGoogle Scholar
- Gygi SP, Rochon Y, Franza BR, Aebersold R: Correlation between protein and mRNA abundance in yeast. Mol Cell Biol 1999, 19: 1720–1730.PubMed CentralPubMedGoogle Scholar
- Ideker T, Thorsson V, Ranish TA, Christmas R, Buhler J, Eng JK, Bumgarner R, Goodlett DR, Aebersold R, Hood L: Integrated genomic and proteomic analyses of a systematically perturbed metabolic network. Science 2001, 292: 929–934. 10.1126/science.292.5518.929View ArticlePubMedGoogle Scholar
- Chen G, Gharib TG, Huang C-C, Taylor JMG, Misek DE, Kardia SLR, Giordano TJ, Iannettoni MD, Orringer MB, Hanash SM, Beer DG: Discordant protein and mRNA expression in lung adenocarcinomas. Mol Cell Proteomics 2002, 1: 304–313. 10.1074/mcp.M200008-MCP200View ArticlePubMedGoogle Scholar
- Lee PS, Shaw LB, Choe LH, Mehra A, Hatzimanikatis V, Lee KH: Insights into the relation between mRNA and protein expression patterns: II. Experimental observations in Escherichia coli. Biotechnol Bioeng 2003, 84: 834–841. 10.1002/bit.10841View ArticlePubMedGoogle Scholar
- Tian Q, Stepaniants SB, Mao M, Weng L, Feetham MC, Doyle MJ, Yi EC, Dai H, Thorsson V, Eng J, Goodlett D, Berger JP, Gunter B, Linseley PS, Stoughton RB, Aebersold R, Collins SJ, Hanlon WA, Hood LE: Integrated genomic and proteomic analyses of gene expression in Mammalian cells. Mol Cell Proteomics 2004, 3: 960–969. 10.1074/mcp.M400055-MCP200View ArticlePubMedGoogle Scholar
- Fare TL, Coffey EM, Dai H, He YD, Kessler DA, Kilian KA, Koch JE, LeProust E, Marton MJ, Meyer MR, Stoughton RB, Tokiwa GY, Wang Y: Effects of atmospheric ozone on microarray data quality. Anal Chem 2003, 75: 4672–4675. 10.1021/ac034241bView ArticlePubMedGoogle Scholar
- Romualdi C, Trevisan S, Celegato B, Costa G, Lanfranchi G: Improved detection of differentially expressed genes in microarray experiments through multiple scanning and image integration. Nucleic Acids Res 2003, 31: e149. 10.1093/nar/gng149PubMed CentralView ArticlePubMedGoogle Scholar
- Garcia de la Nava J, van Hijum S, Trelles O: Saturation and quantization reduction in microarray experiments using two scans at different sensitivities. Stat Appl Genet Mol Biol 2004, 3: Article11.PubMedGoogle Scholar
- Lyng H, Badiee A, Svendsrud DH, Hovig E, Myklebost O, Stokke T: Profound influence of microarray scanner characteristics on gene expression ratios: analysis and procedure for correction. BMC Genomics 2004, 5: 10. 10.1186/1471-2164-5-10PubMed CentralView ArticlePubMedGoogle Scholar
- Khondoker MR, Glasbey CA, Worton BJ: Statistical estimation of gene expression using multiple laser scans of microarrays. Bioinformatics 2006, 22: 215–219. 10.1093/bioinformatics/bti790View ArticlePubMedGoogle Scholar
- Piepho HP, Keller B, Hoecker N, Hochholdinger F: Combining signals from spotted cDNA microarrays obtained at different scanning intensities. Bioinformatics 2006, 22: 802–807. 10.1093/bioinformatics/btk047View ArticlePubMedGoogle Scholar
- Skibbe DS, Wang X, Zhao X, Borsuk LA, Nettleton D, Schnable PS: Scanning microarrays at multiple intensities enhances discovery of differentially expressed genes. Bioinformatics 2006, 22: 1863–1870. 10.1093/bioinformatics/btl270View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.