From Array To High Density Let Robots Do The Work

Low-density arrays in the past used to be produced by simple methods such as slot or spot blotting on filters used with radioactively labeled probes. Nowadays, robotics has taken over, spotting tens of thousands of genes on a single microscope slide with high accuracy. The first robotic arrayers used solid titanium pins that spotted relatively slow and with medium density (spot-spot distance of 300 mM or more). Currently, most people use quill pins, which provide fast printing with high density (spot-spot distance of 200 mM or less). Nevertheless, quill pins have a number of disadvantages that prevent the microarray technique from developing to perfection.1-6-1 As the pins spot the DNA solution, they touch the surface of the slide, which may damage the pins and the slide surface coating where the DNA probe is to be immobilized. As most arrays will be printed using more than one pin, there will be slight differences in spot morphology caused by pin-topin variation. Moreover, the number of slides that can be printed with one dip of a pin in the DNA solution is limited and dependent on the nature of the spotting buffer. Pins may fail during a run by picking up a dust particle or obstruction in the print head causing the whole—or part of the—subarray to drop out. Finally, it appears that thorough washing of the pins between dips is not invariably perfect, causing small amounts of carryover from one spot into the next one.[6]

It is important to notice that when an experimental set of arrays is analyzed, the variation in expression of a single gene across all arrays is analyzed, but not the variation of expression within an array. This implies that localization, pin-to-pin variation, or dye effects do not necessarily need to be corrected for as long as they are reproducible across an experimental set of arrays.

Most of these disadvantages have been overcome by noncontact printers, which are in development. Such printers use ''ink jet'' technology to deposit microdrops on a slide without touching it. Its tips can aspirate enough DNA solution to print thousands of spots, so all arrays within one print run will have identical quality. As the tips do not touch the glass surface, pin-to-pin variation is mostly eliminated and spot morphology is very even. In addition, ink jet technology allows detection of malfunctioning tips using a camera. Afterward, missing spots can be inserted, resulting in 100% complete arrays (Fig. 1).

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Fig. 1 Part of a microarray printed using the SpotArray Enterprise piezoelectric noncontact printer (PerkinElmer) showing complete arrays with very even spot morphology. The array has been constructed using the rat 5 K oligolibrary from Sigma/Compugen (www.cgen.com) on CodeLink slides. (From Ref. [7].) Target labeling, automated hybridization (Hybstation12; Perkin Elmer), and scanning were performed according to the protocols described on our website (www.vumc.nl/microarrays). Samples were from rat brain cerebellum (Cy3) and rat brain cortex (Cy5). (View this art in color at www.dekker.com.)

Finally, noncontact printing provides much more flexibility in designing arrays. It is much easier, for instance, to create replicates by set, rather than consecutively on the array, which introduces more statistical power to the array result as localization effects can be ruled out. In addition, the possibility to print a series of small arrays on one slide aids in the cost-effectiveness needed for diagnostic tests.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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