Environmental Engineering Reference
In-Depth Information
which bind in turn to an RNA-induced silencing complex (RISC). RISC then uses one strand
of siRNA to bind to single-stranded RNA (ssRNA) molecules of complementary sequence,
typically messenger RNAs (mRNAs), and cleave them. Because such mRNAs become
untranslatable once cleaved, their expression is effectively blocked.
Several methods have been developed for delivery of siRNAs to industrial cells: the
chemical or enzymatic synthesis of siRNAs followed by transfection into the target cells,
which is rapid and well-suited to preliminary experiments but is expensive and achieves only
transient interference; the incorporation of siRNA sequences into a DNA plasmid, which also
requires transfection but achieves stable interference once the vector is integrated into the
host genome; and incorporation of siRNA sequences into viral vectors, which can achieve
stable interference and can propagate themselves among target cells but possess potential
biohazard risks [10, 11].
2.4. Microarray Analysis
Essential to the control of gene expression in complicated pathways, such as those
involving the transport of mRNA and/or polypeptides among eukaryotic organelles for
processing, cofactor insertion, and folding, is often the understanding of expression patterns
of the multiple genes involved in the pathway. A tremendous advance in these efforts has
been realized recently in the form of gene microarray technology.
Microarrays are ordered sets of DNA molecules of known sequence applied, often
robotically, in a grid of tiny spots onto a glass slide coated with an organic compound such as
aminosilane to enhance DNA binding. Hundreds to thousands of distinct DNA molecules
may be present in a single microarray, or gene chip, and are most often prepared in one of two
ways: first, by a photolithographic process in which single-stranded oligonucleotides of
unique, desired sequence are synthesized directly on the slide, most often employed in
industry, or second, by physically attaching DNA fragments-such as polymerase chain
reaction (PCR)-amplified genomic library clones-to the solid substrate, used almost
exclusively in academic research. While the former allows higher density of features
(>280,000 on a 1 .28x1 .28-cm array) and elimination of the need to generate cloned DNA or
PCR products, the latter has lower cost and greater flexibility [12].
For analysis of gene expression patterns in eukaryotes, messenger RNA is collected from
cells of interest under two (or more) conditions of interest. Each sample is then reverse-
transcribed into complementary DNA (cDNA) using nucleotides labeled with contrasting
fluorescent dyes. The cyanine dyes Cy3 and Cy5 are popular, although over 70 different dyes
are available [13]. In prokaryotes, a collection of mRNA is impractical due to the absence of
polyadenylation; therefore, total RNA must be collected and labeled by covalent linkage or by
using labeled, but random oligonucleotide primers during reverse transcription.
On printed (non-photolithographic) DNA microarrays, relative transcript abundance is
then measured by hybridizing cDNA samples to the microarray simultaneously and
determining the fluorescence ratio, revealing binding of homologous sequences, for each spot
on the array.
On photolithographic oligonucleotide arrays, in contrast, multiple probes from the same
single gene of interest, each with a corresponding mismatch probe that serves as internal
control, as well as known amounts of labeled transcripts for genes that serve as internal
standards, are hybridized simultaneously to the microarray to enable quantitation of transcript
abundance.
Search WWH ::




Custom Search