- Methodology article
- Open Access
Utilization of a labeled tracking oligonucleotide for visualization and quality control of spotted 70-mer arrays
- Martin J Hessner1, 2Email author,
- Vineet K Singh†3,
- Xujing Wang1, 2,
- Shehnaz Khan2,
- Michael R Tschannen2 and
- Thomas C Zahrt3
© Hessner et al; licensee BioMed Central Ltd. 2004
- Received: 05 December 2003
- Accepted: 09 February 2004
- Published: 09 February 2004
Spotted 70-mer oligonucleotide arrays offer potentially greater specificity and an alternative to expensive cDNA library maintenance and amplification. Since microarray fabrication is a considerable source of data variance, we previously directly tagged cDNA probes with a third fluorophore for prehybridization quality control. Fluorescently modifying oligonucleotide sets is cost prohibitive, therefore, a co-spotted Staphylococcus aureus-specific fluorescein-labeled "tracking" oligonucleotide is described to monitor fabrication variables of a Mycobacterium tuberculosis oligonucleotide microarray.
Significantly (p < 0.01) improved DNA retention was achieved printing in 15% DMSO/1.5 M betaine compared to the vendor recommended buffers. Introduction of tracking oligonucleotide did not effect hybridization efficiency or introduce ratio measurement bias in hybridizations between M. tuberculosis H37Rv and M. tuberculosis mprA. Linearity between the mean log Cy3/Cy5 ratios of genes differentially expressed from arrays either possessing or lacking the tracking oligonucleotide was observed (R2 = 0.90, p < 0.05) and there were no significant differences in Pearson's correlation coefficients of ratio data between replicates possessing (0.72 ± 0.07), replicates lacking (0.74 ± 0.10), or replicates with and without (0.70 ± 0.04) the tracking oligonucleotide. ANOVA analysis confirmed the tracking oligonucleotide introduced no bias. Titrating target-specific oligonucleotide (40 μM to 0.78 μM) in the presence of 0.5 μM tracking oligonucleotide, revealed a fluorescein fluorescence inversely related to target-specific oligonucleotide molarity, making tracking oligonucleotide signal useful for quality control measurements and differentiating false negatives (synthesis failures and mechanical misses) from true negatives (no gene expression).
This novel approach enables prehybridization array visualization for spotted oligonucleotide arrays and sets the stage for more sophisticated slide qualification and data filtering applications.
- Spotted oligonucleotide arrays
- gene expression analysis
Historically, the investigation of genetic alterations has focused on the study of single genes. In recent years, development of DNA microarrays has allowed researchers to study the complete genome of an organism and profile transcriptional expression patterns of up to tens of thousands of genes in a single experiment [1–3]. Consequently, this technology is already providing important insights into the biological properties of various organisms, making it a mainstream component of biomedical research [3–10].
DNA microarrays are currently based on one of two general fabrication formats: (i) light-directed in vitro synthesized oligonucleotide arrays (Affymetrix) [11, 12], or (ii) spotted cDNA or oligonucleotide arrays . While both platforms allow researchers to compare gene expression profiles, each format offers unique advantages and disadvantages. In situ synthesized oligonucleotide arrays offered by Affymetrix are part of a turn-key system that includes both the DNA microarray platform and the corresponding bioinformatic tools. Consequently, investigators can rapidly begin gene expression studies using "off the shelf" reagents and optimized protocols. However, the system and reagents are expensive, thereby limiting the extent of necessary technical and biological replication . In addition, Affymetrix-based arrays cannot be fabricated in the academic laboratory, and offer less flexibility in their design and content compared with spotted cDNA- or oligonucleotide-based arrays. In contrast, spotted cDNA- or oligonucleotide-based arrays utilize high-speed robotics to mechanically or piezoelectrically  deposit small volumes of desired DNA probes onto solid support surfaces. Although substantial optimization of the fabrication process by the user laboratory is required, mechanically spotted arrays can be fabricated "in house", are highly flexible in their design and content, and can be manufactured in reasonably large numbers providing a marked cost savings over commercial array platforms.
Spotted oligonucleotide arrays exhibit a number of advantages over cDNA arrays. For example, oligonucleotides can be synthesized such that homologous sequences between genes can be excluded, thereby enhancing specificity. In addition, a given gene can be represented by a set of different oligonucleotides targeting different regions or exons, allowing for the detection of splice variants, or discrimination of closely related genes, strains, or species. Finally, oligonucleotides can be purchased "ready-to-print", by-passing the labor-intensive probe preparation steps required for utilization of cDNA-based microarrays.
A substantial limitation in the utilization of spotted cDNA- or oligonucleotide-based arrays are their susceptibility to quality control issues, resulting primarily from variable DNA probe deposition and retention on the solid support surfaces. To minimize variations using this fabrication platform, a number of approaches have been described that allow direct visualization of array integrity following printing and blocking procedures. Commonly used methods include the staining of microarrays with DNA-binding fluorescent dyes, or the hybridization of "universal" targets (i.e. random 9-mers) to the spotted DNA elements [15, 16]. While these techniques provide useful information regarding the physical characteristics of the array, its integrity may be compromised during subsequent de-staining or stripping procedures required prior to hybridization of labeled targets . Consequently, investigators typically only examine one or a few representative slides to access the quality of a printed batch.
Previously, we have reported the development and use of a novel three-color cDNA array platform that allows immobilized probes to be directly visualized [17–19]. Utilizing this format, oligonucleotide primers used to amplify cDNA targets are labeled at their 5' end with fluorescein, a dye compatible with commonly used cyanine labeling dyes and confocal laser scanners possessing narrow bandwidths [18, 20]. Element/array morphology, surface DNA deposition/retention, and surface background can be monitored on each slide. Thus, in our laboratory, all cDNA arrays are imaged for quality control prior to hybridization, maximizing the use of quality arrays for subsequent experimental procedures. It is likely that many or all of the benefits to using a directly-coupled fluorophore are also applicable to oligonucleotide-based microarrays; however, synthesis costs make this approach unfeasible. In this report, we describe the use and evaluation of a Staphylococcus aureus-specific fluorescein-labeled 70-mer "tracking" oligonucleotide as a third-color quality control measure of a Mycobacterium tuberculosis-specific oligonucleotide-based microarray.
Rationale and design for tracking oligonucleotide
Utilization of oligonucleotide-based DNA microarrays is limited by the inability to monitor quality control for every array prior to hybridization. If costs were no consideration, it would be ideal to label each oligonucleotide in a given microarray set with a third fluorescent dye so that array fabrication variables could be directly monitored, analogous to the direct labeling methodology we have utilized with cDNA-based microarrays [18, 19]. However, since this practice is currently cost-prohibitive, we reasoned that introduction of a third-color "tracking" oligonucleotide into the printing buffer at low molarity would allow printing fidelity of a target-specific oligonucleotide set to be indirectly monitored. A M. tuberculosis oligonucleotide probe set consisting of 4,269 70-mer oligonucleotides (Operon) was chosen for these studies as our laboratory is currently conducting expression profiling studies on M. tuberculosis strains (Singh and Zahrt, unpublished data).
Probe retention studies
Observation of competitive binding between the tracking and target-specific 70-mers
Introduction of tracking oligonucleotide does not impact Cy3/Cy5 ratio measurement
Analysis of Variance (ANOVA) introduced through the addition of tracking oligonucleotide .
Degrees of Freedom
Prob > F
Quality control and data filtering using the tracking oligonucleotide
This sets the stage for more sophisticated data filtering, however, the signal intensity derived from the tracking oligonucleotide will vary as a function of the binding capacity of the slide, which is influenced by many variables including slide coating chemistry, slide coating lot, printing conditions (temperature and humidity), as well as post-processing variables. Therefore, the binding capacity of the array must first be established using the tracking oligonucleotide alone, to identify the maximum fluorescein intensity for the slide. Quality slides with high retention capacities will have high intensities, whereas low quality/retention slides will not. Likewise, it is also necessary to establish for each slide the fluorescein intensity obtained when co-spotting 0.5 μM tracking oligonucleotide with target-specific oligonucleotide at its proper 40 μM concentration, which is accomplished with a dilution series of positive controls, ie quantified, high synthesis quality oligonucleotides, in known molar ratio with tracking oligonucleotide printed on each array, such as those in Figure 2. The thousands target-specific elements of the array, which include a range of high to low synthesis yield as well as mechanical misses, can now be gauged against these positive and the negative controls.
We have previously shown for cDNA arrays that hybridized data quality is highly dependent on the amount of immobilized probe , and this variable can be controlled using directly labeled probe material and filtering those spots that do not meet defined intensity thresholds. Our current efforts include defining the lowest amount of support-bound target oligonucleotide necessary to obtain reliable ratio measurements, however there are a number of issues to consider. First, cDNA probes typically average about 1 kb in length, which is more than 10-fold the size of a 70-mer oligonucleotide. Therefore it is reasonable to assume that a given area of glass support will bind >10-fold the number of copies of a 70-mer oligonucleotide versus a 1 kb double-stranded PCR product and if ratio data quality is dictated by the number of support-bound copies of a probe sequence, it will be easier to achieve this threshold with spotted oligonucleotides. The second consideration is the signal dynamic range of the tracking oligonucleotide and whether or not data impacting differences in bound target-specific oligonucleotide (aside from synthesis failures) can be measured with this approach. We are currently investigating these issues with the objective of defining the threshold of support-bound probe necessary for reliable gene expression measurements so that these compromising elements can be filtered from data sets.
Variability of microarray data can arise from both biological and technical sources. In this report, use of a fluoresceinated tracking oligonucleotide for quality control purposes has been described. This three-color microarray methodology can be utilized for pre-hybridization quality assessment , to facilitate automated post-hybridization image analysis , as well as post-hybridization data filtering . The ability to perform QC filtering on printed slides allows identification of potentially problematic slides containing low amounts of probe available for hybridization or high variation in spot morphology/deposition across the array.
Mycobacterium tuberculosis 70-mer oligonucleotide set
The Operon (Alameda, CA) 4,269 Mycobacterium tuberculosis 70-mer oligonucleotide probe set was printed on in-house poly-L-lysine coated slides prepared as previously described . Oligonucleotide probes present in this collection were designed from the genomic sequence of M. tuberculosis strain H37Rv and M. tuberculosis strain CDC1551, and include an additional 25 negative and 12 positive oligonucleotide controls. Lyophilized probes were shipped in 384-well micro titer plates. Upon arrival, probes were resuspended to a final concentration of 80 μM in deionized water and resuspended by incubation overnight at 4°C with gentle shaking. Probes were diluted 2-fold into one of three 2 × printing buffers: 6x SSC (s aline s odium c itrate), 100% DMSO (d i m ethyl s ulfo xide), or 30% DMSO containing 3.0 M betaine, thereby generating oligonucleotide sets at a final concentration of 40 μM. When required, a Staphylococcus aureus-specific 70-mer "tracking" oligonucleotide synthesized with a 5'-fluorescein tag was added to the printing buffer at a final molar ratio of 1:80 relative to the M. tuberculosis-specific oligonucleotides.
Printing was performed with a GeneMachines Omni Grid printer (San Carlos, CA) using Telechem International SMP3 pins (Sunnyvale, CA) at 40% humidity and 22°C. To control pin contact force and duration, the instrument was set with the following Z motion parameters, velocity: 7 cm/sec, acceleration: 100 cm/sec2, deceleration: 100 cm/sec2. Following printing, oligonucleotides were UV cross-linked to slides and processed using the previously described nonaqueous blocking protocol . Image files on all arrays were collected before hybridization using excitation and emission spectra specific for fluorescein using a ScanArray 5000 (GSI Lumonics, Billerica, MA).
Generation of third-color Staphylococcus aureus-specific 70-mer oligonucleotide and 5'-Cy5-labeled M. tuberculosis-specific 70-mer oligonucleotides
A 5'-fluoresceinated 70-mer oligonucleotide [5'-(FITC)-ATGAAGAAACTATATACATCTTAT GGCACTTATGGATTTTTACATCAGATAAAAATCAATAACCCGACCC-3'] was generated to the S. aureus response regulator trap and used as a third-color tracking oligonucleotide in the M. tuberculosis 70-mer array. This gene has been shown to be specific to S. aureus , and exhibits no homology to any gene in the M. tuberculosis genome (data not shown). For some experiments, two 5'-Cy5 labeled 70-mer oligonucleotides (Operon) were generated to the aceA (Rv0467) [5'-Cy5-GGATCAACAACGCACTGCAGCGCGCCGACCAGATCGCCAAGATCG AGGGCGATACTTCGGTGGAGAACTG-3'] and sodA (Rv3846) [5'-Cy5-CAAGCTGCTGAT ATTCCAGGTTTACGACCACCAGACGAACTTCCCGCTAGGCATTGTTCCGCTGCTGCG-3'] genes of M. tuberculosis H37Rv. These genes are expressed during growth of M. tuberculosis in laboratory medium in vitro [28, 29].
Growth of Mycobacterium tuberculosis H37Rv and preparation of DNA or RNA
M. tuberculosis H37Rv and the isogenic mprA mutant  were grown at 37°C to an A600 ~ 1.2 in Middlebrook 7H9 broth (Difco, Sparks, MD) supplemented with 10% (vol/vol) albumin-dextrose-catalase (Difco, Sparks, MD) and 0.05% (vol/vol) Tween 80 (Sigma, St. Louis, MO). DNA was prepared from M. tuberculosis as previously described . For preparation of RNA, bacterial cells from a 50.0 ml culture were transferred to a conical tube and concentrated by centrifugation. Cells were resuspended in Tel-Test RNA-Bee (Friendswood, TX) and mechanically lysed using a bead beater (BioSpec Products, Bartlesville, OK) as described . Total RNA was chloroform extracted and precipitated by the addition of isopropanol. The resulting RNA pellet was resuspended in DEPC-treated water and treated with DNase I (Ambion Inc., Austin, TX) to remove contaminating DNA, and re-purified by affinity chromatography using RNeasy purification columns (Qiagen, Valencia, CA). To reduce ribosomal RNA content, 10 μg of DNAse I-treated total RNA was enriched for mRNA using the MICROBExpress™ bacterial mRNA purification kit (Ambion).
Labeling and hybridization of M. tuberculosis RNA or DNA
Enriched mRNA or PCR-amplified DNA was used as templates for labeling reactions microarray hybridization experiments. Test hybridizations utilized aceA (Rv0467) and sodA (Rv3846) Cy 3-labeld PCR products amplified from genomic M. tuberculosis H37Rv DNA using primers [aceAF (5'-GATCCAGCAGGAATGGGACAC-3') and aceAR (5'-CAGACTAGTGGAACTGGCCCTCT-3')] and primers [sodAF (5'-GCCAGACCTGGACTGGGACTA-3') and sodAR (5'-CCGAATATCAACCCCTTGGTCT-3')]. For heterotypic hybridizations, labeled cDNAs were synthesized using SuperScript™ III Reverse Transcriptase (Invitrogen, Carlsbad, CA), a cocktail of synthetically generated decamers , and Cy3- or Cy5-dCTP (Amersham, Piscataway, NJ). Cy3- and Cy5-labeled templates were purified using a Qiagen MinElute kit, and concentrated using Amicon YM 30 columns. The concentrated mixed probes were added to a solution containing 50% formamide, 0.1% SDS, and 5X SSC. Hybridization of M. tuberculosis array slides were performed in sealed humid hybridization cassettes for 16–20 h at 42°C. All slides were pre-hybridized with 0.1% BSA solution in 5X SSC and 0.1% SDS for 30 min at 42°C. Following hybridization, array slides were washed at room temperature for 1 minute each in 2X SSC/0.1% SDS, 1X SSC, 0.2X SSC, and 0.05X SSC and then dried by centrifugation. Following hybridization reactions, arrays were scanned using excitation and emission spectra specific for Cy3 and Cy5. Scanning was performed using a ScanArray 5000 (GSI Lumonics, Billerica, MA).
Data analyses and statistics
Microarray images were processed with Matarray, which has been extended to process three images simultaneously, in particular utilizing the advantages of the pre-hybridization FITC image to assist signal segmentation and quantification [20, 24]. Data filtering and normalization were performed utilizing the quality score q com defined in Matarray . Statistical analysis was performed in Matlab using its statistical toolbox and modules developed in house.
M.J.H is supported by a National Institute of Biomedical Imaging and Bioengineering Grant (EB001421) and by a special fund from the Children's Hospital of Wisconsin Foundation. T.C.Z. is supported by the National Institutes of Health (AI51669). We thank the Clement J. Zablocki VA Medical Center (Milwaukee, WI) for utilization of their BSL-3 bio-containment laboratory and Shuang Jia, M.S., for data preparation.
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