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Numerous factors critically influence array quality and the results obtained upon microarray hybridization and scanning. These factors can be broken down into several general areas: purity and integrity of the material to be printed on the microarray slide, printing of the microarray slide, RNA sample quality (purity, integrity, and labeling), hybridization conditions, and the microarray scanning process (scanner settings and calibration).
Corning® UltraGAPS™ Slides and Pronto!™ Universal Hybridization Kits and Reagents provide users with carefully controlled slides and reagents for the printing and hybridization of printed DNA arrays for gene expression. This control allows for unmatched reproducibility and sensitivity with the ability to measure as little as 1 copy per cell and the ability to obtain inter-slide CV’s of less than 10%. Corning® UltraGAPS™ Microarray Slides are aminosilane-coated glass surfaces. Each slide lot is tested using both physical and functional quality control checks, providing assurance of cleanliness, high surface stability, uniformity and DNA-binding capacity. Pronto!™ Universal Hybridization Kits and Reagents, optimized specifically for use with UltraGAPS™ Slides, are also put through numerous tests to assure users of less lot to lot variability than ever possible with other commercial or homemade systems. Microarrayers now benefit from Corning’s years of experience with microarray printing and hybridization.
The following Troubleshooting Guide covers concerns commonly encountered during the microarraying process.
Common questions and issues associated with microarraying
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Section 1: Microarray Printing
A. General Recommendations: the Printing Process
Spot size and integrity are functions of a number of variables. The chemistry and process in manufacturing UltraGAPS™ slides and Pronto!™ Universal Spotting Solution control the following spot size and spot integrity variables:
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Composition of spotting medium
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Slide surface chemistry
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Slide surface hydrophobicity
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Slide surface cleanliness and integrity
Printing variables unrelated to slide or reagent composition and discussed below are:
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Purity and concentration of cDNA or oligonucleotide material to be printed
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Pin diameter and arrayer settings for pin spacing and dwell time
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Printing environmental conditions
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Relative humidity
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Temperature
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Dust
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Operator handling
Purification and concentration of DNA
To assure that the spotted DNA is clean, of consistent quality and concentration, and the correct sequence, the following steps are required:
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Remove primers and unincorporated nucleotides by filtration (Corning® FiltrEX™ Glass Fiber Plates catalog numbers 3510, 3511, 3533 or PVDF filter plates -catalog numbers 3504, 3531) or column chromatography
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Confirm that each sample is a single PCR product by gel electrophoresis
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Sequence verify samples
For best sensitivity and consistency across the array, measure the concentration of DNA in each well of the source plate before printing. This can be done using a Corning® 96, 96 Half-Area or 384 well UV plate (catalog numbers 3635, 3675, 3679) to quantify spectrophotometric absorbance at A260.
Pin diameter and arrayer settings for pin spacing and dwell time
The ideal spot size depends on the array density one is trying to achieve and the instruments available. Microarray scanners in use today have a maximum resolution of 5 to 10 microns per pixel. Minimally, spots need to be 10 pixels in diameter to provide reliable data. Therefore, when using a 5µm setting, spots must be at least 50µm in size. For a 10µm setting, spots must be greater than 100µm in diameter. It is important to choose the most appropriate pin diameter and pitch (distance between spots) for the desired feature density. The combination of Corning® UltraGAPS™ Slides and Corning® Pronto!™ Universal Spotting Solution results in moderately sized spots and optimal surface coverage. Spot size is dependent on DNA concentration and must be optimized empirically. Optimize pins, pin spacing, and dwell time for each array type by following instructions provided by the printer manufacturer.
Printing environment
The printing environment should be free of dust particles, and kept at a temperature of 20° to 22°C with relative humidity between 45 and 55%
Operator handling
Spurious fluorescence can come from several areas related to slide handling. Both fingerprints and powder from powdered gloves fluoresce in a hybridized microarray. Powder-free gloves should be worn while handling the slides. While a scanner with a confocal lens will be able to avoid adding spurious fluorescence from the back of a slide to spot fluorescent values, CCD camera based scanners do not have that ability. When placing a slide or printed array on a surface, be sure that the surface is clean.
Recommended quality checks for each print run
To determine DNA retention for each batch of printed slides, use a stain such as such as Syto® 61 (by Molecular Probes). This not only confirms DNA retention, but also reveals drop outs and demonstrates spot morphology.
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B. Visual troubleshooting: Printing Issues
Issue: Irregular Spot Morphology and Position
 | Figure 1 - Click for larger image |
Cause: Bent or broken printing pins (Figure 1)
Solution:
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Check physical integrity of the printing pins regularly using a microscope.
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Replace bent or broken pins.
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Replace pins exceeding their useful life – follow manufacturer’s recommendations.
Cause: Build-up of salts and other materials due to sub-optimal washing of printing pins during and after print run (Figures 1 & 2)
Solution:
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Clean printing pins thoroughly after each run – observe manufacturer’s recommendations.
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Optimize pin washing and drying steps of printing program for application at hand.
Cause: Universal Spotting Solution volume differences among wells of a source plate due to “thirsty” pins (Figure 2)
Solution:
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Identify and replace “thirsty” pins.
Comments: Well volume which decreases more quickly in a few wells may indicate that the printing pin visiting those wells is taking up more of the solution than other pins, hence the term “thirsty pin”.
Cause: Printing solution volume differences among wells of a source plate due to evaporation (Figure 2)
Solution:
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Optimize printing humidity.
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Reduce number of freeze thaw cycles.
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Keep print run as short as possible.
Comments: Pronto!™ Universal Spotting Solution is designed to reduce the amount of evaporation and therefore reduce the influence of evaporation on source plate well volumes.
Cause: Addition of detergents or contaminants such as polysaccharides to Pronto!™ Universal Spotting Solution
Solution:
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Use Pronto!™ Universal Spotting Solution as provided
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Do not add detergents or other chemicals to the Universal Spotting Solution.
Comments: Pronto!™ Universal Spotting Solution is quality checked and ready to use as a 1X solution. Detergents and contaminants reduce DNA binding to the UltraGAPS™ surface.
 | Figure 3 - Click for larger image |
Cause: Presence of particulates on the surface of the slide or on the pins(Figure 3)
Solution:
Remove potential sources of surface particulates after package opening. Potential sources include:
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use of powdered gloves while handling slides
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placement of the arrayer near ventilation ducts
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dusty printing area
Comments: While a formal clean-room environment is not generally required, printing should take place in a clean area.
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Issue: Doughnut Shaped Spots
Cause: Contact-point deposition (CPD) due to slide surface damage (Figure 4)
Solution:
The occurrence and negative effects of CPD can be minimized by:
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maintaining printing chamber relative humidity above 45%
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reducing the strike force of the printing pins
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reducing particles in the printing environment and slide handling
Comments: Contact-point deposition (CPD) is a phenomenon involving the anchoring of the liquid contained in a drying droplet onto surface irregularities. Reducing pin strike force minimizes potential physical damage to the aminosilane-coated substrate and the formation of deep anchoring sites for the spotted liquid.
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Issue: Spots with “Comets” or “Tadpoles”
Cause: Loosely bound DNA reattaches to adjacent reactive areas on the slide surface to form comets (Figure 5)
Solution:
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Reduce DNA concentration in the printing solution.
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Optimize DNA concentration for application at hand
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Increase energy input during immobilization of the spotted DNA (UV cross-linking or baking).
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Dry arrays by incubation at 65°C for 5 to 10 minutes immediately before immersion in the pre-hybridization buffer.
Comments: Upon contact with hybridization solutions, loosely bound DNA lifts and reattaches to reactive areas adjacent to the spot. This problem is detected after hybridization and is most visible around spots representing highly expressed genes.
As little as 0.1 mg/mL(~20 µM) of double-stranded DNA or 0.5 mg/mL of 50-mer oligonucleotides can successfully be used for printing.
Increasing energy (increasing UV, baking, or drying after printing) reduces the reactivity of the unused surface before it is exposed to subsequent processing steps. For long oligonucleotides, 600 mJ UV energy is recommended for efficient crosslinking.
Cause: Printed areas not completely dry before beginning hybridization process. (Figure 6)
Solution: Dry array completely before continuing on to hybridizations
Comments: Undried DNA can “drift” from the printed spot resulting in thin tails and uninterpretable data.
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Issue: “Black Holes”
Cause: Very low expressors surrounded by background fluorescence
Solution: Depending upon the appearance of the background around the black hole, refer to appropriate background solutions.
Comments: Black holes appear as dark spots within the background field. These spots have the expected size, shape, and placement of printed spots.
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Issue: DNA Content Carryover (Cross-Talk) During Printing
Cause: Incomplete washing of printer pins between samples.
Solution: Optimize pin wash cycles during printing to make sure no carry over takes place.
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Section 2: Microarray Backgrounds – the Hybridization Process
A. General Recommendations: Microarray backgrounds - Hybridization
At best, a slide with a high background produces a slide that is visually unpleasant. At worst, high background obscures low expressors and the opportunity to fully utilize information present on the slide. Careful attention to following the Pronto!™ protocol brings slide backgrounds to the lowest and most even values possible, providing unparalleled sensitivity and visually pleasing slides for publication.
Several important variables related to hybridization reagents affect final array results and have been optimized in the Pronto!™ Reagent System for use with UltraGAPS™ Slides, including:
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Pre-Soak Solution (Universal kit only)
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Pre-Hybridization Solutions
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Hybridization Solutions
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Wash Solutions
These solutions act to:
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Dramatically reduce autofluorescence from oxidation of the slide surface, DNA Universal Spotting Solution and DNA content
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Effectively block the reactivity of the UltraGAPS™ surface from background caused by nonspecific binding of:
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loosely bound and unbound printed material
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repetitive sequences in labeled cDNA derived from mammalian genomes
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labeled cDNA
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Effectively wash off unbound labeled cDNA
Important variables affecting final array results unrelated to printing slides and the hybridization reagents and discussed below include:
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Fluorescent labeling of cDNA and subsequent removal of unincorporated dyes
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Handling of fluorescent dyes both before and after incorporation into cDNA
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Proper humidified environment
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Slide Handling
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Placement of the coverslip
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Removal of the coverslip
 | Figure 7 - Click for larger image |
Fluorescent labeling of cDNA
It is well worth the time and effort to carefully prepare and quality check the labeled cDNA before application to the printed array. Figure 7 demonstrates the necessity for accurate quantification of labeled cDNA yield and cyanine-dNTP incorporation. As little as a 2-fold excess of labeled cDNA leads to higher background and the false-positive scoring of a bacterial negative control feature. Application of a constant amount of labeled cDNA to each hybridization leads to more reproducible gene expression profiles and reduced variation across experiments. Complete cDNA labeling instructions can be found in the Pronto!™ Universal Hybridization Kits and Reagents operating instructions. Important quality checks include:
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RNA Integrity
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A260/280 ratio between 1.8 and 2.0
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Purity and integrity analysis by gel electrophoresis – no smearing or extra bands
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Labeled dye incorporation
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Frequency of Incorporation (FOI) calculation:
FOI= pmol incorporated dye x 324.5 (the average molar mass of a deoxynucleotide base)/ng cDNA in sample recommended 15 to 45 labeled nucleotides per 1,000 cDNA nucleotides, depending on RNA source (total RNA or mRNA) and Cy® dye used where:
Amount of labeled cDNA (ng) = A260 × 37 × total volume (µl). Note: for a solution with an A 260 = 1.0, the following approximations hold:
1 A260 unit of dsDNA = 50 ug/ml
1 A260 unit of ssDNA = 37 ug/ml
1 A260 unit of ssRNA = 40 ug/ml
For Cy®3 dye: pmol of dye incorporated = A550 x total volume (µl) / 0.15.
For Cy®5 dye: pmol of dye incorporated = A650 × total volume (µl) / 0.25.
Note: 0.15 and 0.25 are derived from the extinction coefficients for Cy®3 and Cy®5 dyes, respectively.
Fluorescent dye handling
Fluorescent dyes are very susceptible to chemical breakdown due to environmental effects. The best known effect is photobleaching, caused by exposure to ambient light. Also important to consider are air pollutants which enhance oxidation of the dyes. Keep dyes out of direct light, cover tubes with aluminum foil, and leave capped as much as possible. The UltraGAPS™ orange slide storage container is ideal for hybridized array storage as the high technology plastic keeps out light wavelengths that excite (and therefore breakdown) fluorescent dyes.
Proper humidification
A humidified environment during hybridization incubation is required to prevent the labeled cDNA from drying out. Corning recommends the Corning® Hybridization Chamber (catalog number 2551 or 40080). This reusable chamber provides a watertight seal for use in either a water bath or incubation oven and contains humidity wells for the addition of buffer.
Slide Handling
For researchers using a glass coverslip, a certain amount of care and practice is required in the addition of the labeled cDNA to the slide and coverslip placement to prevent smearing, scratching, and fluorescent artifacts. For new users, practice with blank slides and buffer will quickly provide proficiency and good results. The steps to proper technique are:
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Pipette labeled cDNA in several drops down the center of the slide
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In one even motion, slowly lower the coverslip onto the slide and direct any air bubbles to the leading edge of the buffer
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Do Not Attempt to move the coverslip once it is on the array. This will cause smearing of the spots or scratches on the surface, resulting in uninterpretable data
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Small bubbles will frequently dissipate during hybridization
Removal of the coverslip
Remove the coverslip by placing the coverslipped array into the first wash solution. The wash solution will work its way between the array and the coverslip and the coverslip will fall away from the array. Do not attempt to physically remove the coverslip as removal may cause scratches. Do not use tweezers near the array surface.
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B. Visual Troubleshooting: Microarray backgrounds - Hybridization
Issue: High and/or Uneven Background Levels
 | Figure 8 - Click for larger image |
Cause: Presence of contaminating materials, unincorporated fluorescent nucleotides, and aggregated labeled DNA in the hybridization mix (Figure 8)
Solution:
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Prepare hybridization buffers shortly before use.
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Use 18 mega-ohm Milli-Q™ deionized water, or its equivalent for reagent preparation.
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Remove unincorporated nucleotides from the labeled DNA (most effectively by column chromatography).
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Boil the hybridization mix before application to the array, do not place on ice
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Purify mRNA prior to reverse transcription (optional).
Cause: Improper storage of slides after opening foil pouch (Figure 8)
Solution:
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Reseal unused slides in UltraGAPS™ storage container immediately after removing the slides to be used for printing.
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Keep unused slides sealed and provide with a dry environment at normal ambient temperature.
Comments: Use slides from properly stored packages within 7 days of opening. The UltraGAPS™ storage container was specifically designed for storage of UltraGAPS™ Slides and is ideal for storage of UltraGAPS™ Slides before and after printing.
Cause: Deposition of volatile organics onto the slide surface (Figure 8)
Solution:
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Check for volatile organics entering the printing environment
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Check for possible build-up of organic materials used to lubricate the moving parts on the arraying instruments.
Comments: These materials may be present in the printing environment or elsewhere in the laboratory, and may themselves be fluorescent or have high affinity for fluorescent contaminants. Some arraying instruments use organics that may become volatile during the printing process. Mass spectrometers can also generate aerosolized organics.
Cause: RNA contaminated with polysaccharides, proteins or DNA
Solution: Ensure starting material for generation of labeled cDNA is as pure as possible.
Comments: If the RNA used to generate the labeled cDNA is not free of contaminants, the resultant hybridization will be poor.
 | Figure 9 - Click for larger image |
Cause: Incomplete slide washing.
(Figure 9)
Solution:
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Use 18 mega-ohm Milli-Q™ deionized water, or its equivalent, for wash solution preparation.
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Carry out washing steps in the order and at the stringencies outlined in the Pronto!™ protocol booklet.
Comments: Washing is one of the most critical steps in obtaining consistently low backgrounds. Incomplete washing causes deposition of salts and fluorescent materials that may cover all or part of the array. Also, it is important to not let the slide dry during the washing process.
 | Figure 10 - Click for larger image |
Cause: Evaporation of buffers or wash solution on the slide surface (Figure 10)
Solution: Do Not allow slides to dry out once the hybridization process is started
Comments: As buffers evaporate, salts dry on the surface in somewhat concentric circles.
 | Figure 11 - Click for larger image |
Cause: Improperly washed coverslips (Figure 11)
Solution: Wash coverslips with water and then with ethanol. Allow the coverslips to dry completely in a dust-free environment prior to placing them on the arrays.
 | Figure 12 - Click for larger image |
Cause: Scanner stage misalignment
(Figure 12)
Solution: Check for misalignment by reversing the orientation of the array on the scanning stage and rescan.
Comments: If the direction of the change in signal strength is reversed, adjustments to the scanner need to be made by a service representative. Some scanners do not focus steadily as the arrays are scanned, leading to gradual changes in the spot signal intensities across the arrays.
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Issue: Smearing Across Large Portions of the Array
 | Figure 13 - Click for larger image |
Cause: Improperly applied coverslip, coverslip slid across or pressed onto array (Figure 13)
Solution:
Be particularly careful when handling arrays after the coverslip has been placed.
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Do not place coverslip onto array with heavy pressure.
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Make sure that the coverslip does not move during application or hybridization.
Comments: Application of the coverslip can take some time to learn. Practice using plain glass slides and buffer until the fluid motion is learned.
Cause: Array surface comes in contact with another slide or walls of prehyb or wash container
Solution: Make sure that the array surface does not come in contact with surfaces other than a properly applied coverslip.
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Issue: Weak Spot Signal Intensities
Cause: Poor retention of spotted DNA
Solution:
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Optimize printing conditions for each array type.
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Use Pronto!™ Universal Spotting Solution as provided, do not dilute, add detergents or other chemicals.
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Use slides before their “best use by date”.
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Use Pronto!™ Universal Spotting Solution before the “best use by date”.
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Follow crosslinking instructions carefully.
Comments: Long oligonucleotides require more energy to efficiently crosslink to UltraGAPS™ surface. 600mJ UV energy is recommended for long oligonucleotides.
Poor retention of spotted DNA can lead to low signal levels and under-estimation of transcript abundance. Fluorescent dye staining (such as Syto® 61 by Molecular Probes) can be used to determine if poor retention is the issue behind weak spot signals.
Although Corning® UltraGAPS™ Slides and Pronto!™ Reagents can perform well beyond the “best use by” date indicated on the label, it is best practice to use slides and reagents before these dates.
Cause: RNA template contaminated with DNA
Solution: Check RNA purity and integrity before labeling
Comments: DNA contamination can be determined by gel electrophoresis performed before RNA labeling, and estimated by measuring the A260/A280 ratio.
Cause: Inefficient or excessive incorporation of fluorescent dNTPs into cDNA
Solution: Make labeled cDNA to incorporate fluorescent dNTPs at the rate of between 15 and 45 per thousand nucleotides of cDNA
Comments: The frequency of incorporation of fluorescent dNTPs should be checked before labeled cDNA is used. Follow the methods described in the Pronto!™ Istruction Manual. With either Cy-dye, if the frequency of incorporation (FOI) is too high (>45 Cy-dCTPs/kb) or too low (<15 Cy-dCTPs/kb), a decrease in signal has been seen.
 | Figure 14 |
Cause: Cy® dye photobleaching
Solution:
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Protect labeled cDNAs and hybridized arrays from light.
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Wrap tubes containing fluorescent reagents in aluminum foil.
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Hybridize, wash, and store arrays away from strong light sources.
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Cover arrays with opaque material as much as possible.
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Avoid multiple scans of arrays.
Comments: Fluorescent dyes such as those typically used for microarray work are photosensitive. Cy®
dye strength can decrease drastically if exposed to brightly-lit environments. Importantly, the graph above (Figure 14) demonstrates that Cy®5 dye degradation can lead to incorrect interpretation of differential gene expression (i.e., at Scan 1, Cy®5 dye signal is higher than Cy®3 dye, and at Scan 5, the ratio is reversed).
The new UltraGAPS™ orange slide cases provide low transmittance (500-700nm) and help protect Cy®3 dye and Cy®5 dye from photo bleaching hybridized arrays.
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Issue: Regularly Shaped Dark Region of Scanned Array
 | Figure 15 - Click for larger image |
Cause: Air bubbles trapped under coverslip (Figure 15)
Solution: Carefully apply coverslip to the array after addition of the labeled cDNA to the array surface.
Comments: Using buffer and empty slides, practice placing coverslips until comfortably proficient at the maneuver. Slowly lower the coverslip onto the slide and direct any air bubbles to the leading edge of the buffer. When placing a coverslip onto a printed array, if an air bubble becomes trapped in the hybridization buffer, no attempt should be made to remove it, as this may scratch the array surface. Sometimes air bubbles move to the edge and dissipate during hybridization. Avoiding the formation of bubbles when resuspending labeled cDNA in hybridization buffer will also help to minimize entrapment of bubbles on the array.
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Issue: Miscellaneous Fluorescent Oddities
 | Figure 16 - Click for larger image |
Cause: Labeled cDNA not evenly distributed on array (Figure 16)
Solution:
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Pipette 4 to 6 drops along the middle line of the array.
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Immediately apply the coverslip to the array, avoiding bubbles as much as possible.
Comments: Application of the labeled cDNA at only one end of an array (top or bottom) almost always results in images containing areas of high background near the point of application.
Cause: Labeled cDNA dries out at coverslip edges (Figure 16)
Solution:
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Humidify hybridization environment.
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Increase hybridization buffer volume.
Comment: The air volume surrounding the array must be saturated with water vapor. Otherwise, as the array is brought to incubation temperature, water from the labeled cDNA will be drawn into the air within the Hybridization Chamber, thereby decreasing its volume. Use of Corning® Hybridization Chambers (catalog number 2551 or 40080) and the filling of each humidification well with 10 to 12 µl of water is strongly recommended.
Cause: Powder from gloves and other environmental particulates (dust)
Solution:
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Wear powder-free gloves during array printing and hybridizing.
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Perform arraying under environmentally controlled conditions, where the presence of dust particles is minimized.
Comments: Powder and dust can be strongly fluorescent and produce spots of various shapes and sizes that interfere with data interpretation.
 | Figure 17 - Click for larger image |
Cause: Scratches on slide surface due to scraping during slide processing (Figure 17)
Solution:
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Allow coverslip to slide off during the first wash.
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Take care not to scrape the surface of the slide when manipulating it with forceps.
Comments: Do not forcibly remove the coverslip from the array surface.
 | Figure 18 - Click for larger image |
Cause: Corning® Hybridization Chamber humidity well(s) overfilled (Figure 18)
Solution: Do not pipette more than 10 to 12 µL water into each hybridization chamber humidity well.
Comments: If more than 12 µL of fluid is placed in either humidity well, the top of the slide may come in contact with the large bubble of water, and water may wick between the coverslip and the array surface causing extraneous fluorescence.
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Issue: Spot Signals Attained When Using Corning Pronto!™ Reagents are Noticeably Different than Signals Attained When Using Homemade Buffers
Cause: Insufficient blocking in homemade buffers to prevent nonspecific hybridization.
Solution: UltraGAPS™ Slides and Pronto!™ Reagents combine to yield more reliable and biologically relevant results.
Comments: The Corning® Pronto!™ Reagent Systems were formulated to eliminate most nonspecific binding during hybridization. Expression patterns using the Pronto!™ Kits have been confirmed by comparison to published Northern Blot data, as well as by Taqman® assays.
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