title: Forschungszentrum Karlsruhe Danio rerio microarray
status: Public on Feb 01 2007
contact.city: Eggenstein-Leopoldshafen
contact.first: Jessica
contact.last: Legradi
contact.state: Baden-Wuerttemberg
contact.address: Hermann-von-Helmholtz-Platz 1
contact.department: Institute of Toxicology and Genomics ITG
contact.country: Germany
contact.institute: Karlsruhe Institute of Technology
organism: Danio rerio
description: The oligos were spotted onto the chip in 4 by 12 blocks where each block is printed by one single pin. A block itself is divided into a subgrid of 27 x 27 spots and each sequence of 16 spots contains oligos from a different library plate. The spots are arranged densely on the array. An optimal spot has a diameter of 100 um and the distance between blocks is 150 um. The library provided by Sigma-Genosys/Compugen comprises 43 plates. A major drawback in the biosensor project is that only oligos from plate 1 to 28 yielded a proper signal. Oligos from the remaining plates 29 to 43 were extended with defect linkers. Hence, they were not able to bind permanently to the chip surface which made them useless for microarray analysis. Although the original library comprises of 16 228 oligonucleotides approximately one third had to be excluded completely from further statistical analysis. An additional plate with oligos from our lab was spotted onto the chip. Arrangement of spots impacts further ,statistical normalisation analysis. In order to increase precision of signals each oligo is spotted twice. In particular metacolumn 3 and 4 are an exact copy of metacolumn 1 and 2 which allows to calculate an average signal for each gene based on two spots. In order to minimize correlation caused by spatial background patterns, the two copies were not placed in adjacent blocks. To establish a thorough quality control of arrays, beta actin and so-called spikes were considered during microarray design. Beta actin is a housekeeping gene which is in constant use to maintain basic cell functions. Therefore, it is expected to express at the same level under different treatment conditions. The corresponding mRNA is present in large numbers so it yields signals of very high intensity. Beta actin is arranged multiple times on the last two metarows of the chip which can be observed as a chessboard-like pattern. Additionally, spikes were included to check the correct labelling of samples.
manufacture_protocol: Printing is performed with the multi-axis Omnigridder 100 spotter1 from Genomic Solutions with a capacity of 100 chips.
  For transferring the sample probes onto the array a printhead and 24 SMP3 pins from TeleChem are used.
  The sample solution is spotted onto 'SurModics 3D-Link' slides which are available as 'CodeLink activated slides' from Amersham Biosciences3. To be more precise, those chips are covered by a 3-D gel matrix build up of a hydrophilic, long chain polymer that contains amine-reactive groups. The polymer is covalently cross-linked to itself and to the surface of the slide. It orients the immobilized oligos and thus holds them away from the surface of the slide. Accordingly, the sample probes have to be extended by an amino linker to stick permanently to the slide. As a consequence, the oligos are more readily available for hybridisation and spacers are not needed any more. In addition, the hydrophilic nature of the polymer provides a passivating effect after the DNA has been immobilized resulting in higher sensitivity and lower background.
  We have used the oligo set from Sigma-Genosys/Compugen containing 16 228 oligos.
  Prerequisites for microarray production include thorough cleaning of the equipment and calibration of the z-axis (vertical) movement of each pin. The latter is crucial as the pins are supposed to touch the slide surface only gently. Imprecise calibration of the pin movement possibly either fails to deliver the spot liquid or it hits and scratches the chip.
  Before starting the actual print, test runs are conducted by spotting 2-3 test chips so that calibration and quality of pins can be refined. Optimal spotting results require a constant temperature of 18 C and humidity of 60%.
  One of 43 sample library plates (384-well) is picked up by the server arm and placed in the nest.
  The printhead with its 2 by 12 pins dips into the plate and takes up 24 different sample probes which are spotted simultaneously onto the array.
  Afterwards the pins are sequentially dipped into water, dried with pressurised air, and cleaned in a sonicator.
  This washing routine is performed three times before the next 24 samples are taken up by the pins.
  After 16 cycles all samples of the 384-well have been spotted and the next plate is processed. The overall printing process of 100 chips runs for about one day.