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FAQ - Questions around Microarraying

Can I print on both sides of the slides?

With the exception of Slide H, P, NC and HiSens all other NEXTERION® slides are coated on both sides and hence can be printed on either side.
The SCHOTT slide boxes are manufactured from a special plastic that minimises the outgassing of plasticizes, as these can have an adverse affect on performance of the slide coating. However, there can still be some slight effect, so we recommend that you do not print on the outer surface of the two end slides that face the side of the box. The drawing illustrates this for slides stored in a 25-slide mailer box.

Which side of the HiSens slide should I print on?

How to identify the correct side:

There is a narrow transparent section at one end of the slide. This strip contains a small tab / mark (red arrow on figure below). If you orientate the tab so that it is in the lower right hand corner, you are looking at the surface for printing.

What parameters affect the printing process?

The printing is affected by the:
    1) Surface (hydrophobicity and uniformity)
    2) Probe (quality and concentration)
    3) Print buffer (composition)
    4) Printing environment (humidity, temperature and cleanliness)
    5) Printing hardware (pin type, quality and tip diameter, dwell time)
1) Surface
Microarray surfaces from SCHOTT are produced under controlled clean room conditions and provide a very uniform coating, which allow for a reliable probe deposition. However, they differ in their chemistry (active or passive) and to some extent in their hydrophobicity, which could be relevant if array density is a crucial factor.
2) Probe
Probe molecules should have a consistent quality and concentration, to allow a meaningful data comparison.
3) Print buffer
The choice of the print buffer is very important, as this influences the choice of the substrate chemistry, spot morphology and size, signal intensity, storage and stabilization of probe molecules.
Some print buffers like DMSO, that are well suited for Aminosilane surfaces, don’t perform as well on other surfaces. For example on Epoxysilane or Aldehydesilane, DMSO produces spots that are very small.
SCHOTT NEXTERION® provides optimal print buffers for the respective surfaces and probe molecules (see table below).
NEXTERION® Coating Spotting Blocking Hybridisation
Spotting Buffer Low evaporation buffer
NEXTERION® Spot PB(Protein applications)
H NEXTERION® Spot PB * * NEXTERION® Hyb (DNA applications)
P NEXTERION® Spot PB * * NEXTERION® Hyb (DNA applications)
* No pre-made NEXTERION® reagent available. Please check the appropriate slide protocol for alternative recommendations.
Spot sizes can be adjusted by adding the appropriate amount of detergent to the print buffer. Another useful consideration in the beginning is the compatibility of the print buffer with the envisaged printing speed, printer type and desired storage conditions. If printing runs are planned for several hours or even days it will be useful to use low evaporation buffers like 50% DMSO or NEXTERION® Spot A HD, as probe concentration remains more constant compared to aqueous print buffers. Printers that allow for humidity adjustment, source plate cooling or source plate covering during the print run are more flexible in terms of print buffer choice, because evaporation can be better controlled. On the other hand aqueous print buffers allow rapid evaporation in e.g. speed vac and storage of probes in a dry status, which is essential to some users.
4) Environmental printing conditions
We recommend printing at room temperatures (15-25°C) and at relative humidities between 40 and 65% (humidity should be stable during the whole printing run). We would not recommend to exceed printing humidities of 70% which might be attractive to control evaporation. Moreover, it is advisable to print in a clean environment, preferable under clean room conditions to avoid contamination of dust, which can affect printing.
5) Printing hardware
Other options to control spot size are dwell time (the longer the bigger the spot) and more importantly the choice of an appropriate pin. Tip diameter can vary between ca. 50 – several hundred µm. This is especially important if one intend to print high density arrays. The lower limit of spot size should be around 50µm, at least when standard scanners are used, which are able to scan images at a resolution down to 4 - 5 µm. In this case at least 10 pixel per spot are analyzable. With standard slides and conventional printers array densities can be up to 50,000 – 75,000 features per slide.

How can I check the quality of the printed slide?

1. You can use dyes like SYBR Green, TOPRO-3 and SYTO 59, which basically tell you whether and how much DNA is bound to the surface.
2. Alternatively you can use labeled random oligos (like Cy3 or Cy5 panomers), which will give you the same information as in 1) but will also indicate if the DNA in the spot is available for hybridization.

I am using split pins with a pin diameter of 75 µm and getting spot sizes of 50 to 60 µm with Next Spot. How can I increase the spot size?

This spot size is what we often observe with this type (diameter) of split pins. To increase the spot size detergents may be added to the spotting solution. To find the optimal spotting solution, it is advisable to test two or three different detergent concentrations, for example with SDS (0.005 - 0.05 or Triton X100 (0.01 - 0.1 % final concentration) or sarcosyl (0.00025 - 0.001 % final concentration in the print buffer). If you intend to print proteins, then using 0.005 – 0.02 % Tween20 might be a better option as Tween20 will have a less effect on protein structure.
NEXTERION® Spot is supplied as a twofold (2x) concentrated solution. The former NEXTERION® Spot II and III and AM II are also 2x concentrated and can be prepared directly from the 2x NEXTERION® Spot.

Trouble Shooting Guide with Images

Big spots, merged spots
Merged Spots
When a pin is dipped into sample, there is always some sample that sticks to the outside of the pin. This so called “rush” effect may result in bigger spots or even fused/merged spots. Removal of the liquid on the outside of the tip will produce better spots. This is typically done by pre-printing (pre-blotting) about 10-100 times (depending on the viscosity of the print buffer) on pre-blot slides before moving the pin set to the "real" slides/substrates. Alternatively, the amount of liquid on the outside of the pin may be reduced by either withdrawing the pins more slowly from the source plate or redipping the pin briefly into the sample.
If that does not help then either increasing the pitch or decreasing the spot size (by e.g. use of smaller pins) should be considered.
Irregular spot shape
Check if the pins are clean and are delivering solution onto the slide surface. If the pins are clogged or empty, the tip of the pins may only leave irregular shaped marks/droplets on the slide.
Irregular spot morphology - “fried egg” effect
This effect is more frequently seen with biphasic print buffers. Due to the different solubility of the probe molecule, this phenomenon can occur. High evaporation rates are known to promote this phenomenon as well. In this case, increasing the humidity (during print runs or afterwards) can help to improve spot morphology.
None or too little probe delivery after printing a few slides
(Biorobotics printer, Biorobotics split pins type 2500):
Change printer settings: Modify the software settings so that the pins dip slowly into the source plates, as this allows the pin reservoir to fill up fully without the risk of bubble formation.
Black holes or negative spots
"Black holes" or negative spots as a general phenomenon on a slide indicate impure or degraded starting material or problems with the target e.g. low dye incorporation or dye oxidation.
If Black holes only occur occasionally then the printed probe has no or very low binding affinity to the target molecules.
However, in both cases the higher background around the spots indicate a suboptimal blocking or pre-hybridization step.

Comet tailing
Comet tails are formed when unbound nucleic acids bind to the activated slide surface around the spot during the slide processing steps i.e. during the washing steps before blocking.

Several users have reported that fast and vigorous shaking during the first washing/blocking steps also helps to reduce comet tail formation.

Aminosilane coated slides
After spotting Aminosilane coated slides, such as NEXTERION® Slide AStar and A+, it is important to covalently immobilize the bound molecules by baking or UV-crosslinking and to inactivate the slide surface by blocking to prevent re-binding. Additionally lowering the concentration of nucleic acids in the spotting solution will help to reduce the excess of unbound molecules.

All other NEXTERION® coated slides
For active slide chemistries like Slide E, AL, P and H, the best approach would be to wash slides directly after immobilization with a specific chemical blocking solution. This will help to block/inactivate the surface immediately and prevent excess probe material from binding to the surface. Additionally lowering the concentration of nucleic acids in the spotting solution will help to reduce the excess of unbound molecules.

Doughnut spot morphology
In general, doughnuts are most commonly seen with contact printers. One possible cause can be a high evaporation rate during spotting, as the spotted solution has not enough time to spread evenly. Increasing the relative humidity, or reducing the printing temperature during spotting to about 50 – 65 % rel. humidity (at appr. 20 to 23°C) may reduce this effect. An alternative is to use a spotting solution with a reduced evaporation rate such as 3xSSC containing 1.5 M betaine for Slide E, or a lower concentration of DMSO for Slide AStar, and A+.

However, there are also other alternatives for reducing doughnuts:
  • Reduce the force of the pins for touching the surface. Use a "soft touch mode" in combination with the pin speed of 4 cm/sec.
  • The spot morphology of NEXTERION® Slide E could be significantly improved by treating the slides after the printing process in a humidity chamber with a relative humidity greater than 90% at room temperature.

How should I prepare a suitable humidity chamber?

A convenient and simple option is to use a sealable plastic container that has a tray for slides, and below the tray a large petri-dish. This petri-dish is filled with steaming hot water 30 minutes before the slides are placed into the container. Within this 30 minutes the atmosphere is saturated with water and the temperature is around room temperature. The humidity chamber is ready for the slides once this environment is achieved.

Do I need to increase oligonucleotide concentration if using non-modified rather than modified oligonucleotides for spotting on NEXTERION® Slide E?

No. This is because at the recommended concentrations the modified oligonucleotides are already in excess compared to the binding capacity of the slide surface.

What is the maximum number of spots that can be printed on a standard NEXTERION® slide?

Many factors such as printing pins, equipment and printing conditions affect the spot density on a slide. However, with a printing area of 72 x 22 mm at least 50000 spots should be achievable.

How large is the actual printing area on the slides?

The recommended printing area is an area of 72 x 22 mm for slides without barcode, and 64 x 22 mm for slides with barcode.

I'm buying a UV Crosslinker to attach my DNA probes to either NEXTERION® Slide A+ or Slide A*. The NEXTERION® protocol specifies the total energy, e.g. 250 mJ, but not the UV wavelength.

Shorter wavelengths (254 nm) of UV light will cross-link DNA by covalent interaction with the primary amines.
Suitable commercial UV crosslinkers include:
    BioLink BLX Multipurpose UV Crosslinker - 254, 312 or 365 nm
    Stratalinker 2400 - 254 nm
    Hoefer UV Crosslinker - 254

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