18.1 Transcriptomics

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Table 18.1 Typical features of microarrays

Application

Capture element

Sample

Genomics

ESTs

DNA

Transcriptomics

cDNA

mRNA

Proteomics

Antibodies

Proteins

Metabolomics

Various

Various

sequentially. The parent classical assay is the Northern blot. ² Microarrays consist of

a two-dimensional array, typically a few square millimetres in overall area, of more

or less contiguous patches, the area of each patch being a few square micrometres (or

less) and each patch on the array having a different chemical composition. Typical

microarrays are assembled from one type of substance (e.g., nucleic acid oligomers).

In use, the array is flooded with the sample whose composition one is trying to

elucidate. ³ After some time has elapsed, the array is scanned to determine which

patches have captured something from the sample. It is, of course, essential that each

patch should be addressable, in the sense that the composition of each individual

patch is known or traceable. Hence, a photomicrograph of the array after exposure

to the analyte should allow one to determine which substances have been captured

from the sample.

Table 18.1 summarizes some features of microarrays. In more detail, the protocol

for a microarray assay would typically involve the following steps:

Array preparation. The chip should be designed on the basis of what one is looking

for. Each gene of interest should be represented by at least one, or preferably more,

unique subsequences. ⁴ Once the set of sequences has been selected, there are two

main approaches to transfer them to the chip:

1. Heteroöligomers complementary to the mRNA of interest are assembled from

successive monomers using microfabrication technology; for example, ⁵ photoac-

tivatable nucleic acid monomers are prepared. Exposure through a mask, or with

a laser scanner, activates those patches selected to receive, say, G. After exposure

to light, the array is then flooded with G. Then the array is exposed to a differ-

ent pattern and again flooded (with a different base), and so on. This technology

2 Northern blotting allows detection of specific RNA sequences. RNA is fractionated by agarose gel

electrophoresis, followed by transfer (blotting) to a membrane support, followed by hybridization

with known DNA or RNA probes that are radioactively or fluorescently labelled to facilitate their

detection. The technique can be thought of as a variant of Southern blotting, in which specific DNA

sequences from a sample are probed in a similar fashion.

3 If one is trying to determine whether certain genes are present in a bacterial culture (for example),

the array would be coated with patches of complementary nucleic acid sequences. The DNA is

extracted from the bacteria, subjected to some rudimentary purification, separated into single strands,

and usually cut into fragments with restriction enzymes before pouring over the microarray.

4 See Chumakov et al. (2005) for a discussion of design principles.

5 Fodor et al. (1991).