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A) The steps required in a microarray experiment from sample preparation to analyses. RT, Reverse transcriptase. For details see text. Briefly, samples
are prepared and cDNA is created through reverse transcriptase. The fluorescent label is added either in the RT step or in an additional step after
amplification, if present. The labeled samples are then mixed with a hybridization solution that contains light detergents, blocking agents (such as COT1
DNA, salmon sperm DNA, calf thymus DNA, PolyA or PolyT), along with other stabilizers. The mix is denatured and added to a pinhole in a microarray,
which can be a gene chip (holes in the back) or a glass microarray. The holes are sealed and the microarray hybridized, either in a hybridization oven,
(mixed by rotation), or in a mixer, (mixed by alternating pressure at the pinholes). After an overnight hybridization, all nonspecific binding is washed off.
Source: Methods of Molecular Analysis, The Basic Science of Oncology, 5e
The microarray is dried and scanned in a special machine where a laser excites the dye and a detector measures its emission. The intensities of the
Citation:
Tannock
RP, Bristow
RG, Harrington
L. The Basic
Science
Oncology, 5e;
2016
Available at:
http://mhmedical.com/
features (several
pixels
makeIF,a Hill
feature)
are quantified
and normalized
(see
text). of(Reproduced
with
permission
from
Jacopo Werther/Wikimedia
Accessed:
May
03,
2017
Commons.) B) The output from a typical microarray experiment, a hierarchical clustering of cDNA microarray data obtained from 9 primary laryngeal
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© 2017 McGraw-Hill
All rights
tumors. Results
were visualized
using TreeEducation.
View software,
andreserved
include the dendrogram (clustering of samples) and the clustering of gene expression,
based on genomic similarity. Tree View represents the 946 genes that best distinguish these 2 groups of samples. Genes whose expression is higher in