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FAQ - Questions around Microarraying
Hybridization

Which factors affect hybridization?

Hybridization can be affected by several factors: Sequence and quality of DNA Hybridization temperature Hybridization buffer (ionic strength) Agitation rate Hybridization time Concentration of DNA Surface charge Linker length and flexibility In contrast to hybridization carried out in solution, on a microarray slide one of the binding partners is fixed at the surface for hybridization. As a result of this it is advisable to add a linker to short oligo sequences to allow the maximum accessibility.

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What is the difference between NEXTERION® Hyb (order code: 1066075) and NEXTERION® Oligo Hyb (order code: 1116890)?

Both NEXTERION® Hyb Buffers were especially developed to provide the customer with an optimized and ready-to-use buffer to improve the reproducibility of microarray experiments. In contrast to "NEXTERION® Hyb", the "NEXTERION® Oligo Hyb" buffer contains formamide and competitor DNA. Therefore the hybridization temperature should be adjusted to 42°C. The "NEXTERION® Hyb" buffer does not contain formamide, and should be used at higher hybridization temperatures between 55°C and 65°C.

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How can I formulate my own hybridization buffer?

We recommend using 3x to 5x SSC including 0.1% SDS with or without formamide and competitor DNA.

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Does the pre-hybridization buffer need to contain formamide?

There is no need to put formamide in the pre-hybridization solution because stringency only matters during the actual hybridization. However adding formamide to the prehyb solution means that the same solution can be used for both the pre-hybridization and hybridization.

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What can I check if there is no signal on the substrate after hybridization?

No signal could be caused by a number of factors. Basically either something has gone wrong during the slide processing, or there is a quality issue with the labeled target. To rule out processing errors, labeled oligos should be printed alongside the probes, and the slide should be scanned after each main processing step, such as printing, blocking and hybridization. The target material could be checked in various ways such as: Spot check with Cy3 or Cy5 panomers Gel electrophoresis UV/Vis spectrometry

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What are the causes of an uneven hybridization pattern across an array?

An uneven hybridization could be due to one of the following causes Uneven distribution of the target sample over the array (image 1) Presence of air bubbles in the hybridization solution (image 2) This can be an issue if very low volumes are used. Therefore careful pipetting, and precise sealing of the chambers are important ways to avoid this problem. Partial drying of the hybridization solution during incubation (image 3) This can also be a problem if very low volumes are used. Therefore the precise sealing of the chambers, or incubation in humidity chambers should help to overcome this issue. An external factor like fluorescent contamination from a new gasket or chamber of a hybridization stations An hybridization time that is too short Using buffers that are too viscous. None or too little agitation.

Image 1 Irregular target distribution

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Image 2 Air bubbles in hybridization solution

Image 3 Dried hybridization solution

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How can I decrease high background fluorescence observed after hybridization?

A high background fluorescence after hybridization could be caused by one the following: Poor quality of labeled target (Image below) Check the quality of your labeled target with an appropriate method like UV-spectroscopy or gel-electrophoresis if possible. Ensure complete removal of unbound fluorescent dyes or dye-coupled oligonucleotides after the labeling reaction. Inadequate blocking. To avoid this problem carefully follow the respective slide processing protocol regarding the blocking and washing steps. Drying of the hybridization solution during the incubation. This might indicate a faulty valve in the hyb station or insufficient humidity during the manual hybridization steps. Insufficient washing after hybridization might lead to an increased background fluorescence. To avoid insufficient washing after hybridization: Remove the coverslip in the washing buffer by dipping the slide into the first washing buffer and agitating until the coverslip falls off. Transfer the slide into new buffer. Use enough washing buffer and agitation during washing, do not re-use washing buffer. Do not allow the slides to dry between washing steps. Prepare the next washing buffer beforehand in a new beaker and transfer slide quickly. Quickly dry the slides after the last washing step (for instance by centrifugation in a 50 ml falcon tube), to avoid drying stains.

Image 4 poor target quality

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Can SCHOTT recommend any alternatives to the Cy5 dye?

We use ATTO 647N from ATTO-TEC in our lab as Cy5 replacement. https://www.atto-tec.com/attotecshop/product_info.php?info=p114_ATTO-647N.html&XTCsid=ejjspmkvatbrv13lk8iopd7nn2 Another alternative we use is DY-647 from Dyomics. The dye is much more stable than Cy5, but not as stable as ATTO 647N. http://www.dyomics.com/dy-647.html