Sequence-dependent fluorescence of cyanine dyes on microarrays

Cy3 and Cy5 are among the most commonly used oligonucleotide labeling molecules. Studies of nucleic acid structure and dynamics use these dyes, and they are ubiquitous in microarray experiments. They are sensitive to their environment and have higher quantum yield when bound to DNA. The fluorescent intensity of terminal cyanine dyes is also known to be significantly dependent on the base sequence of the oligonucleotide. We have developed a very precise and high-throughput method to evaluate the sequence dependence of oligonucleotide labeling dyes using microarrays and have applied the method to Cy3 and Cy5. We used light-directed in-situ synthesis of terminally-labeled microarrays to determine the fluorescence intensity of each dye on all 1024 possible 5'-labeled 5-mers. Their intensity is sensitive to all five bases. Their fluorescence is higher with 5' guanines, and adenines in subsequent positions. Cytosine suppresses fluorescence. Intensity falls by half over the range of all 5-mers for Cy3, and two-thirds for Cy5. Labeling with 5'-biotin-streptavidin-Cy3/-Cy5 gives a completely different sequence dependence and greatly reduces fluorescence compared with direct terminal labeling.     

A highly fluorescent DNA toolkit: synthesis and properties of oligonucleotides containing new Cy3, Cy5 and Cy3B monomers

Cy3B is an extremely bright and stable fluorescent dye, which is only available for coupling to nucleic acids post-synthetically. This severely limits its use in the fields of genomics, biology and nanotechnology. We have optimized the synthesis of Cy3B, and for the first time produced a diverse range of Cy3B monomers for use in solid-phase oligonucleotide synthesis. This molecular toolkit includes phosphoramidite monomers with Cy3B linked to deoxyribose, to the 5-position of thymine, and to a hexynyl linker, in addition to an oligonucleotide synthesis resin in which Cy3B is linked to deoxyribose. These monomers have been used to incorporate single and multiple Cy3B units into oligonucleotides internally and at both termini. Cy3B Taqman probes, Scorpions and HyBeacons have been synthesized and used successfully in mutation detection, and a dual Cy3B Molecular Beacon was synthesized and found to be superior to the corresponding Cy3B/DABCYL Beacon. Attachment of Cy3, Cy3B and Cy5 to the 5-position of thymidine by an ethynyl linker enabled the synthesis of an oligonucleotide FRET system. The rigid linker between the dye and nucleobase minimizes dye-dye and dye-DNA interactions and reduces fluorescence quenching. These reagents open up new future applications of Cy3B, including more sensitive single-molecule and cell-imaging studies.     

Effects of a modified dye-labeled nucleotide spacer arm on incorporation by thermophilic DNA polymerases

The ability of eight commercially available thermophilic DNA polymerases to sequentially incorporate fluorescently labeled nucleotides sequentially was analyzed by a gel based primer extension assay. Cy5-dUTP or a variant nucleotide in which the linker had been lengthened by 14 atoms between the dye and the nucleobase were compared. We found that the Cy5-dUTP with a longer linker resulted in longer primer extension lengths. Furthermore, some of the assayed polymerases are capable of extending the primer to the full or near full length of 30 nucleotides using dye-labeled nucleotides exclusively.     

Microarray scanner calibration curves: characteristics and implications

Background

Microarray-based measurement of mRNA abundance assumes a linear relationship between the fluorescence intensity and the dye concentration. In reality, however, the calibration curve can be nonlinear.

Results

By scanning a microarray scanner calibration slide containing known concentrations of fluorescent dyes under 18 PMT gains, we were able to evaluate the differences in calibration characteristics of Cy5 and Cy3. First, the calibration curve for the same dye under the same PMT gain is nonlinear at both the high and low intensity ends. Second, the degree of nonlinearity of the calibration curve depends on the PMT gain. Third, the two PMTs (for Cy5 and Cy3) behave differently even under the same gain. Fourth, the background intensity for the Cy3 channel is higher than that for the Cy5 channel. The impact of such characteristics on the accuracy and reproducibility of measured mRNA abundance and the calculated ratios was demonstrated. Combined with simulation results, we provided explanations to the existence of ratio underestimation, intensity-dependence of ratio bias, and anti-correlation of ratios in dye-swap replicates. We further demonstrated that although Lowess normalization effectively eliminates the intensity-dependence of ratio bias, the systematic deviation from true ratios largely remained. A method of calculating ratios based on concentrations estimated from the calibration curves was proposed for correcting ratio bias.

Conclusion

It is preferable to scan microarray slides at fixed, optimal gain settings under which the linearity between concentration and intensity is maximized. Although normalization methods improve reproducibility of microarray measurements, they appear less effective in improving accuracy.

     

Effects of a Modified Dye-Labeled Nucleotide Spacer Arm on Incorporation by Thermophilic DNA Polymerases

The ability of eight commercially available thermophilic DNA polymerases to sequentially incorporate fluorescently labeled nucleotides sequentially was analyzed by a gel based primer extension assay. Cy5-dUTP or a variant nucleotide in which the linker had been lengthened by 14 atoms between the dye and the nucleobase were compared. We found that the Cy5-dUTP with a longer linker resulted in longer primer extension lengths. Furthermore, some of the assayed polymerases are capable of extending the primer to the full or near full length of 30 nucleotides using dye-labeled nucleotides exclusively.     

Correcting for gene-specific dye bias in DNA microarrays using the method of maximum likelihood

MOTIVATION: In two-color microarray experiments, well-known differences exist in the labeling and hybridization efficiency of Cy3 and Cy5 dyes. Previous reports have revealed that these differences can vary on a gene-by-gene basis, an effect termed gene-specific dye bias. If uncorrected, this bias can influence the determination of differentially expressed genes. RESULTS: We show that the magnitude of the bias scales multiplicatively with signal intensity and is dependent on which nucleotide has been conjugated to the fluorescent dye. A method is proposed to account for gene-specific dye bias within a maximum-likelihood error modeling framework. Using two different labeling schemes, we show that correcting for gene-specific dye bias results in the superior identification of differentially expressed genes within this framework. Improvement is also possible in related ANOVA approaches. AVAILABILITY: A software implementation of this procedure is freely available at http://cellcircuits.org/VERA     

Analysis of DIGE data using a linear mixed model allowing for protein-specific dye effects

Differential in-gel electrophoresis (DIGE) experiments allow three protein samples to be run per gel. The three samples are labeled with the spectrally resolvable fluorescent dyes, Cy2, Cy3, and Cy5, respectively. Here, we show that protein-specific dye effects exist, and we present a linear mixed model for analysis of DIGE data which takes dye effects into account. A Java implementation of the model, called DIGEanalyzer, is freely available at http://bioinfo.thep.lu.se/digeanalyzer.html. Three DIGE experiments from our laboratory, with 173, 64, and 24 gels, respectively, were used to quantify and verify the dye effects. DeCyder 5.0 and 6.5 were used for spot detection and matching. The fractions of proteins with a statistically significant (0.001 level) dye effect were 19, 34, and 23%, respectively. The fractions of proteins with a dye effect above 1.4-fold change were 1, 4, and 6%, respectively. The median magnitude of the dye effect was 1.07-fold change for Cy5 versus Cy3 and 1.16-fold change for Cy3 versus Cy2. The maximal dye effect was a seven-fold change. The dye effects of spots corresponding to the same protein tend to be similar within each of the three experiments, and to a smaller degree across experiments.     

Comparison of Alexa Fluor<Superscript>®</Superscript> and CyDye<Superscript>™</Superscript> for practical DNA microarray use

Microarrays are a powerful tool for comparison and understanding of gene expression levels in healthy and diseased states. The method relies upon the assumption that signals from microarray features are a reflection of relative gene expression levels of the cell types under investigation. It has previously been reported that the classical fluorescent dyes used for microarray technology, Cy3 and Cy5, are not ideal due to the decreased stability and fluorescence intensity of the Cy5 dye relative to the Cy3, such that dye bias is an accepted phenomena necessitating dye swap experimental protocols and analysis of differential dye affects. The incentive to find new fluorophores is based on alleviating the problem of dye bias through synonymous performance between counterpart dyes. Alexa Fluor 555 and Alexa Fluor 647 are increasingly promoted as replacements for CyDye in microarray experiments. Performance relates to the molecular and steric similarities, which will vary for each new pair of dyes as well as the spectral integrity for the specific application required. Comparative analysis of the performance of these two competitive dye pairs in practical microarray applications is warranted towards this end. The findings of our study showed that both dye pairs were comparable but that conventional CyDye resulted in significantly higher signal intensities (P < 0.05) and signal minus background levels (P < 0.05) with no significant difference in background values (P > 0.05). This translated to greater levels of differential gene expression with CyDye than with the Alexa Fluor counterparts. However, CyDye fluorophores and in particular Cy5, were found to be less photostable over time and following repeated scans in microarray experiments. These results suggest that precautions against potential dye affects will continue to be necessary and that no one dye pair negates this need.     

Phosphorimeters for analysis of decay profiles and real time monitoring of exponential decay and oxygen concentrations

A phosphorimeter which can be assembled at low cost from mainly commercially available components and which has better time resolution, data acquisition rate, sensitivity, and flexibility than commercially available instruments is described. As a phosphorescence analyzer the instrument can measure phosphorescence lifetimes ranging from approximately 30 microseconds to seconds from samples with variable intensity, excitation, and emission spectra and which may follow complex decay behavior. Configured as a phosphorescence monitor it is designed for fast, repetitive calculation of phosphorescence lifetime, assuming single-exponential decay, and can be used to calculate oxygen concentration in biological samples in real time.     

Cowpea mosaic virus nanoscaffold as signal enhancement for DNA microarrays

Previous studies have shown that a functionalized viral nanoparticle can be used as a fluorescent signal-generating element and enhance detection sensitivity for immunoassays and low density microarrays. In this study, we further tested this ability in commercial DNA microarrays, including Affymetrix high density resequencing microarray. Optimum conditions for NeutrAvidin and dye coupling to a double-cysteine mutant of cowpea mosaic virus (CPMV) were found to be comparable to the commonly used streptavidin-phycoerythrin (SAPE) for high density resequencing microarray. A 3-fold signal enhancement in comparison to Cy5-dCTP controls was obtained when using nanoparticles on control scorecard expression microarrays. Hybridization results from commercially available 8000 rat expression arrays indicate an increment of 14% on the detected features when the virus complex was used as the staining reagent in comparison to Cy5-dCTP controls. The current work shows the utility of the CPMV-dye nanoparticles as a detection reagent in well-established detection platforms.     

Statistical design of reverse dye microarrays

MOTIVATION: In cDNA microarray experiments all samples are labelled with either Cy3 dye or Cy5 dye. Certain genes exhibit dye bias-a tendency to bind more efficiently to one of the dyes. The common reference design avoids the problem of dye bias by running all arrays 'forward', so that the samples being compared are always labelled with the same dye. But comparison of samples labelled with different dyes is sometimes of interest. In these situations, it is necessary to run some arrays 'reverse'-with the dye labelling reversed-in order to correct for the dye bias. The design of these experiments will impact one's ability to identify genes that are differentially expressed in different tissues or conditions. We address the design issue of how many specimens are needed, how many forward and reverse labelled arrays to perform, and how to optimally assign Cy3 and Cy5 labels to the specimens. RESULTS: We consider three types of experiments for which some reverse labelling is needed: paired samples, samples from two predefined groups, and reference design data when comparison with the reference is of interest. We present simple probability models for the data, derive optimal estimators for relative gene expression, and compare the efficiency of the estimators for a range of designs. In each case, we present the optimal design and sample size formulas. We show that reverse labelling of individual arrays is generally not required.     

Evaluation of two-dimensional differential gel electrophoresis for proteomic expression analysis of a model breast cancer cell system

The technique of fluorescent two-dimensional (2D) difference gel electrophoresis for differential protein expression analysis has been evaluated using a model breast cancer cell system of ErbB-2 overexpression. Labeling of paired cell lysate samples with N-hydroxy succinimidyl ester-derivatives of fluorescent Cy3 and Cy5 dyes for separation on the same 2D gel enabled quantitative, sensitive, and reproducible differential expression analysis of the cell lines. SyproRuby staining was shown to be a highly sensitive and 2D difference gel electrophoresis-compatible method for post-electrophoretic visualization of proteins, which could then be picked and identified by matrix-assisted laser-desorption ionization mass spectroscopy. Indeed, from these experiments, we have identified multiple proteins that are likely to be involved in ErbB-2-mediated transformation. A triple dye labeling methodology was used to identify proteins differentially expressed in the cell system over a time course of growth factor stimulation. A Cy2-labeled pool of samples was used as a standard with all Cy3- and Cy5-labeled sample pairs to facilitate cross-gel quantitative analysis. DeCyder (Amersham Biosciences, Inc.) software was used to distinguish clear statistical differences in protein expression over time and between the cell lines.     

Pre-labeling of diverse protein samples with a fixed amount of Cy5 for sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis

A pre-labeling protocol based on Cy5 N-hydroxysuccinimide (NHS) ester labeling of proteins has been developed for one-dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis. We show that a fixed amount of sulfonated Cy5 can be used in the labeling reaction to label proteins over a broad concentration range—more than three orders of magnitude. The optimal amount of Cy5 was found to be 50 to 250 pmol in 20 μl using a Tris–HCl labeling buffer at pH 8.7. Labeling protein samples with a fixed amount of dye in this range balances the requirements of sub-nanogram detection sensitivity and low dye-to-protein (D/P) ratios for SDS–PAGE. Simulations of the labeling reaction reproduced experimental observations of both labeling kinetics and D/P ratios. Two-dimensional electrophoresis was used to examine the labeling of proteins in a cell lysate using both sulfonated and non-sulfonated Cy5. For both types of Cy5, we observed efficient labeling across a broad range of molecular weights and isoelectric points.     

Self-self hybridization as an alternative experiment design to dye swap for two-color microarrays

Dye-specific bias effects, commonly observed in the two-color microarray platform, are normally corrected using the dye swap design. This design, however, is relatively expensive and labor-intensive. We propose a self-self hybridization design as an alternative to the dye swap design. In this design, the treated and control samples are labeled with Cy5 and Cy3 (or Cy3 and Cy5), respectively, without dye swap, along with a set of self-self hybridizations on the control sample. We compare this design with the dye swap design through investigation of mouse primary hepatocytes treated with three peroxisome proliferator-activated receptor-alpha (PPARalpha) agonists at three dose levels. Using Agilent's Whole Mouse Genome microarray, differentially expressed genes (DEG) were determined for both the self-self hybridization and dye swap designs. The DEG concordance between the two designs was over 80% across each dose treatment and chemical. Furthermore, 90% of DEG-associated biological pathways were in common between the designs, indicating that biological interpretations would be consistent. The reduced labor and expense for the self-self hybridization design make it an efficient substitute for the dye swap design. For example, in larger toxicogenomic studies, only about half the chips are required for the self-self hybridization design compared to that needed in the dye swap design.     

Possible sources of dye-related signal correlation bias in two-color DNA microarray assays

DNA microarray analyses commonly use two spectrally distinct fluorescent labels to simultaneously compare different mRNA pools. Signal correlation bias currently limits accepted resolution to twofold changes in gene expression. This bias was investigated by (i) examining fluorescence and absorption spectra and changes in relative fluorescence of DNAs labeled with the Cy3, Cy5, Alexa Fluor 555, and Alexa Fluor 647 dyes and by (ii) using homotypic hybridization assays to compare the Cy dye pair with the Alexa Fluor dye pair. Cy3 or Cy5 dye-labeled DNA exhibited reduced fluorescence and absorption anomalies that were eliminated by nuclease treatment, consistent with fluorescence quenching that arises from dye-dye or dye-DNA-dye interactions. Alexa Fluor 555 and Alexa Fluor 647 dye-labeled DNA exhibited little or no such anomalies. In microarray hybridization, the Alexa Fluor dye pair provided higher signal correlation coefficients (R2) than did the Cy dye pair; at the 95% prediction level, a 1.3-fold change in gene expression was significant using the Alexa Fluor dye pair. Lowered signal correlation of the Cy dye pair was associated with high variance in Cy5 dye signals. These results indicate that fluorescence quenching may be a source of signal bias associated with the Cy dye pair.     

The reaction of protein tryptophanyl residues with reduced nicotinamide adenine dinucleotide. Structure of a model adduct.

This paper describes in detail a simple, light producing and handling system which differs greatly from the commercial unit presently used by most investigators, and which overcomes a number of its disadvantages. The apparatus is designed so that it can easily be substituted for the light producing, dispersing, and collimating section of the commercial photoelectric scanners, with no further changes in the commercial scanners except the substitution of optical flats for the collimating and condensing lenses in the bottom and top, respectively, of the rotor chamber. The most outstanding feature of this new system is that the use of a faster monochromator ($\text{f}\text{75.3}$) and a cylindrical lens theoretically increases light intensity by up to 100 times that of the commercial scanner, under otherwise identical conditions. Other advantages include the following: (1) For collecting and collimating light, two pairs of mirrors give a precell optical system focused in the radial direction at all wavelengths of light; (2) these components and the lamp are located on a table outside the centrifuge, so virtually any size of lamp can be used; (3) the entire precell optical system is a self-contained unit which, within reasonable limits, may be moved to any desired location without markedly affecting the quality of the collimated light; and (4) the optical path length has been shortened, providing less dissipation of light energy. The principles behind the selection and design of the key components are discussed. New alignment procedures and apparatus developed to aid in fast, easy, accurate alignment are described and discussed. Several components for use with a system using a computer-controlled stepping motor scanner for collection of data are also described. This system is simple enough and is documented in sufficient detail so that other interested workers, even those with little or no optics experience, can duplicate and use the system.     

Picosecond multiphoton scanning near-field optical microscopy.

We have implemented simultaneous picosecond pulsed two- and three-photon excitation of near-UV and visible absorbing fluorophores in a scanning near-field optical microscope (SNOM). The 1064-nm emission from a pulsed Nd:YVO4 laser was used to excite the visible mitochondrial specific dye MitoTracker Orange CM-H2TMRos or a Cy3-labeled antibody by two-photon excitation, and the UV absorbing DNA dyes DAPI and the bisbenzimidazole BBI-342 by three-photon excitation, in a shared aperture SNOM using uncoated fiber tips. Both organelles in human breast adenocarcinoma cells (MCF 7) and specific protein bands on polytene chromosomes of Drosophila melanogaster doubly labeled with a UV and visible dye were readily imaged without photodamage to the specimens. The fluorescence intensities showed the expected nonlinear dependence on the excitation power over the range of 5-40 mW. An analysis of the dependence of fluorescence intensity on the tip-sample displacement normal to the sample surface revealed a higher-order function for the two-photon excitation compared to the one-photon mode. In addition, the sample photobleaching patterns corresponding to one- and two-photon modes revealed a greater lateral confinement of the excitation in the two-photon case. Thus, as in optical microscopy, two-photon excitation in SNOM is confined to a smaller volume.     

A two-photon and second-harmonic microscope

Two-photon microscopy has revolutionized life sciences by enabling long-term imaging of living preparations in highly scattering tissue while minimizing photodamage. At the same time, commercial two-photon microscopes are expensive and this has prevented the widespread application of this technique to the biological community. As an alternative to commercial systems, we provide an update of our efforts designing custom-built two-photon instruments by modifying the Olympus FluoView laser scanning confocal microscope. With the newer version of our instrument we modulate the intensity of the laser beam in arbitrary spatiotemporal patterns using a Pockels cell and software control over the scanning. We can also perform simultaneous optical imaging and optical stimulation experiments and combine them with second harmonic generation measurements.