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Download e-book for kindle: Cell-Based Microarrays: Review of Applications, Developments by Ella Palmer

By Ella Palmer

This booklet is a evaluate at the evolution of cell-based microarrays and an replace to the author's previous e-book Methods in Molecular Biology: Cell-Based Microarrays. given that their improvement in 2001, cell-based microarrays have complicated significantly to incorporate expression arrays, brief interfering RNA arrays and antibody arrays. the skin used to coat the glass slides has additionally been considerably more desirable to permit non-adherent cells to bind to the arrays.

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5 mL of medium were added to a corner of a rectangular well, whilst avoiding direct application of the cells to the printed array. After an adherence time of 15 min, cells that had not adhered were washed off. This approach resulted in a lower number of cells per spot than usual: For instance, after 48 h of cultivation, 51 ± 3 PC-3 cells were found on a spot with a diameter of 200 μm, and 151 ± 3 cells on a 400 μm spot. Using primary human prostate stromal cells, the counts were even lower, as 21 ± 6 cells were found on a 200 μm spot.

4 23 shRNA-Mediated Knock-Down The application of siRNAs as immediate effectors of knock-down—on a microarray or otherwise—is prone to dilution effects in proliferating cells, as the effective siRNA concentration per cell becomes reduced upon mitosis. A work-around for this problem lies in the constitutive replenishment of siRNAs by the cell, which is accomplished by the delivery of DNA coding for short hairpin RNAs (shRNAs). This approach was independently developed by Greg Hannon’s group [19] and Reuven Agami’s group [20], enabling the establishment of stable knock-down in mammalian cells: Both groups made use of RNA polymerase III promoters, the transcripts of which have defined start and end points.

45] observed a significant reduction in vimentin expression as monitored by immunofluorescence microscopy. 7 Concluding Remarks The chip-based microarrays discussed in this chapter mostly result in a monolayer of cells, of which only those cells that happen to be seeded on a spot take up the genetic information. Thus, the cells are free to leave “their” spot and move around later on. This lack of confinement might be problematic for some sorts of assays, and can be either overcome by blocking the area outside the spots to cell adherence, as shown by Oehmig et al.

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