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Frequently Asked Questions (FAQ)

Welcome to the frequently asked questions (FAQ) section. This list is designed to assist in answering questions related to NEXTERION® microarray products. The list is divided into the following sections: Questions around NEXTERION® products NEXTERION® uncoated glass NEXTERION® aminosilane coated products NEXTERION® aldehydesilane coated products NEXTERION® epoxy coated products NEXTERION® 3-D hydrogel coated products NEXTERION® 3-D polymer coated products NEXTERION® HiSens coated products Miscellaneous Questions related to the microarray process Printing and Immobilization Blocking Hybridization Scanning Microarraying on multi-well formats Image gallery of typical microarray problems If your microarray spots don't appear as they should, have a look at the image gallery of typical problems. Link to the gallery

General questions around NEXTERION® Products

NEXTERION® uncoated glass What is the difference between SCHOTT NEXTERION® Glass B and D? What is the tensile strength of NEXTERION® Glass B and D, how do these values compare to soda lime glass? How does the hardness of Glass B and D compare to Soda lime glass? Has BOROFLOAT® 33 / NEXTERION® Glass B been tested for hydrolytic resistance in accordance with the European Pharmacopeia EP 6 (2010) Chapter 3.2.1 and United States Pharmacopeia USP 32? What is the average surface micro roughness (Ra) of Glass B (BOROFLOAT® 33) and D (D263)? Are the properties of the two Glass D (D263) surfaces identical? NEXTERION® Glass B (BOROFLOAT® 33) is produced by the microfloat process, resulting in a "fire-polished" upper surface, and a lower surface that has been in contact with molten tin. Is it possible to tell the difference between the two surfaces? What is the "flatness" of Glass B (BOROFLOAT® 33)? Can SCHOTT offer any advice on the best method of bonding Glass D (D263) to Polydimethylsiloxane (PDMS)? In the NEXTERION® Glass B specification, a number of spot type defects are listed under "Surface specification". What do these terms mean? Can you certify that NEXTERION® Glass B / BOROFLOAT 33 does not contain any toxic heavy metals? We are using NEXTERION® Glass B / BOROFLOAT® 33 in a diagnostic product / medical device, can you certify that the glass does not contain any animal derived material? Does SCHOTT offer alternative glass types for biological applications? What is the difference between the three levels of cleanliness of the SCHOTT NEXTERION® uncoated slides? What is the refractive index of the glass used to manufacture the NEXTERION® Glass B slides? What is the refractive index of the glass used to manufacture the NEXTERION® Glass D slides? Which excitation and emission wavelengths has the Glass B and D been tested with for auto-fluorescence? What is the transmission / absorbance spectrum of Glass B (BOROFLOAT® 33)

NEXTERION® aminosilane coated products Can SCHOTT recommend any blocking protocols for the NEXTERION® aminosilane slides that do not involve BSA? What is the density of functional groups on the slide surface? For printing DNA oligonucleotides onto NEXTERION® aminosilane slides (Slides A / A+ / A*), your protocols state that I can use 50% DMSO. What should I dilute the DMSO with? 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.

NEXTERION® aldehydesilane coated products What is the density of functional groups on the slide surface?

NEXTERION® epoxysilane coated products What is the density of functional groups on the slide surface?

NEXTERION® 3-D hydrogel coated products Why do you recommend that the Slide H is stored at -20°C? Is this just a precautionary measure or is it based on experiments? What is the thickness and micro-roughness of the SCHOTT NEXTERION® Slide H coating? What solvents can be used in my spotting buffer for printing on NEXTERION® Slide H? If I want to store my NEXTERION® Slide H slides after printing but before hybridization, what are the recommended conditions for protein and DNA arrays? How can I make the deactivation solution for NEXTERION® Slide H? NEXTERION® Slide H has a three dimensional coating. Are there any issues that I need to know about if I scan these slides with a (MDS Axon) GenePix® scanner that has a user-selectable focus position ? I want to print on to NEXTERION® Slide H - Can you recommend a method of preparing the print buffer (150mM sodium phosphate buffer at pH 8.5) ?

NEXTERION® 3-D polymer coated products Currently there are no specific FAQs for this product type

NEXTERION® HiSens coated products Currently there are no specific FAQs for this product type

Miscellaneous What barcode options can SCHOTT offer? What is the location of the black / white laser bonded barcodes on the NEXTERION® slides? Can SCHOTT Microarray Solutions offer custom logos? Will the NEXTERION® slides fit into standard microarray equipment? What is the shelf life of the NEXTERION® coated and uncoated slides? What are the recommended storage conditions for the slides? Will you fax or e-mail me an article from your bibliography? How are the NEXTERION® microarray subtrates packaged? How firmly are the SCHOTT NEXTERION® coatings attached to the glass? What is the density of functional groups on the slide surface? What factors should be considered when selecting dyes? How can I increase the cDNA labelling efficiency when using the aminoallyl-method? What is the optimal labeling ratio for proteins? What factors affect Cy3/Cy5 relation?

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Questions around Microarraying

Printing and Immobilization The printing process accomplishes the delivery of the probes onto the microarray slide and should be as uniform as possible to allow a reliable data generation. During immobilization probes are covalently attached to the surface to allow for subsequent processing. Can I print on both sides of the slides? Which side of the HiSens slide should I print on? What parameters affect the printing process? How can I check the quality of the printed slide? 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? Trouble shooting guide spots Big spots, merged spots Irregular spot shape Irregular spot morphology - “fried egg” effect No or too less probe delivery after printing a few slides Black holes or negative spots Comet tailing Doughnut spot morphology How should I prepare a suitable humidity chamber? For printing DNA oligonucleotides onto NEXTERION® aminosilane slides (Slides A / A+ / A*), your protocols state that I can use 50% DMSO. What should I dilute the DMSO with? 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. Do I need to increase oligonucleotide concentration if using non-modified rather than modified oligonucleotides for spotting on NEXTERION® Slide E? What is the maximum number of spots that can be printed on a standard NEXTERION® slide? How large is the actual printing area on the slides?

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Blocking Blocking is conducted after the immobilization of the probes, and either inactivates the remaining active groups on the surface, or blocks the surface by complete occupation of the remaining binding sites. What is the recommended method of preparing NEXTERION® Block E? What is the pH of the NEXTERION® Block E before, and after adding the required water and hydrochloric acid? How stable is the diluted NEXTERION® Block E? Can I leave out the blocking steps for NEXTERION® Slide AStar and E? I want to store my slides after printing; but before hybridization. Should I store them directly after printing, or after the washing and blocking steps? How can I avoid bleeding spots or comet tails? In the DNA application protocol for NEXTERION® Slide AL, you recommend a "chemical deactivation" step with Sodium borohydride (NaBH4). This does not appear in the protein application protocol, and seems to be replaced by a blocking step. Is the chemical deactivation and blocking the same?

Hybridization The hybridization process should be designed to allow the specific matching of the labeled binding partner, but at the same time avoiding unspecific binding of non-complementary sequences. Moreover, unspecific binding of any other fluorescent compounds to the surface should be minimized to maximise the sensitivity of the experiment. Which factors affect hybridization? What is the difference between NEXTERION® Hyb (order code: 1066075) and NEXTERION® Oligo Hyb (order code: 1116890)? How can I formulate my own hybridization buffer? Does the pre-hybridization buffer need to contain formamide? What can I check if there is no signal on the substrate after hybridization? What are the causes of an uneven hybridization pattern across an array? How can I decrease high background fluorescence observed after hybridization? Can SCHOTT recommend any alternatives to the Cy5 dye?

Scanning Can I use the NEXTERION® HiSens reflective slides in all scanner systems? What should I do if the PerkinElmer ScanArray scanner gives the error message “Slide not recognized” when I try to scan a NEXTERION® Slide MPX?

Microarraying on multi-well formats General Which side of the MPX slide/plate is the coated? What is the recommended minimum distance from the array to the edge of the patterning? What difficulties are caused by printing the arrays too close to the black PTFE well patterning? How can I avoid cross contamination between neighboring wells on the plate? What is the recommended volume that can be added to each well in the MPX-96 superstructure? For the post hyb washing, is it advisable to remove the superstructure? Can SCHOTT provide alternative patterning formats to the 16/48-well MPX slides and 96-well microplate? Can the MPX slides, plates or the superstructures be ordered separately? Over what temperature range can I use the 96-well silicone superstructures? What types of reagents are the 96-well silicone superstructures compatible with? Multi-well slide format Is there a dimensional drawing that indicates the relative positions of the Slide MPX-16 wells when loaded into the MPX-4 tray? We need to determine the row and column locations of the slide's assay wells with respect to the edges of the tray so we can set up our liquid handler.

Multi-well plate format How can I firmly fix the glass plate into the plate holder during printing and scanning? How do I locate the A1 well on the MPX-96 glass plate? Which is the recommended spotting solution for the MPX-96 plates? What is the best way to accurately attach the superstructure to the plate? Are there any commercial available automated plate washers that can be used with the NEXTERION® MPX-96 plate? What is the best approach to remove the superstructure from the plate after the hybridization step? Is there an optimal temperature for removing the superstructure from the glass plate? Can the MPX-96 kit be used more than once? What are the dimensions of the wells on the NEXTERION® MPX-96 plates? What is the recommended minimum and maximum buffer volume that I can add to the wells of the NEXTERION® MPX-96 plate?

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