Biomedical Engineering Reference
In-Depth Information
5'
AAAAAAAAAAAA 3'
mRNA
Reverse
Transcriptase
oligo-dT-T7
T7
TT TTT TTT TTT T
AAAAAAAAAAAA 3'
5
3'
5'
cDNA
TTT TTT
TT
TTT T
T7
DNA
Polymerase
3'
5'
3'
TTT TTT
TT
TTT T
T7
T7
ds-cDNA
5'
AAAAAAAAAAAA
T7
Polymerase
aa-UTP
3'
UUUUUUUUUUUU 5'
3'
UUUUUUUUUUUU 5'
UUUUUUUUUUUU 5'
3'
3'
UUUUUUUUUUUU 5'
aa-cRNA
3'
UUUUUUUUUUUU 5'
3'
UUUUUUUUUUUU 5'
Figure 5.1
Generalized T7-amplification schema. mRNA is reverse transcribed into cDNA. The
oligo-dT primer used for RT also has a T7-promoter sequence appended to it on the 5
end.
After second-strand cDNA synthesis is complete, an artificial gene with a T7-promoter is formed.
Addition of T7-polymerase results in hundreds to thousands of copies of cRNA being generated.
A modified nucleotide such as aminoallyl-UTP can be added during transcription to allow for
subsequent chemical labeling with fluorophores.
inexpensive and highly sensitive. PCR amplification also results in DNA-based amplimers
which are much more stable than their RNA counterparts. In some embodiments of the
methodology, only a small portion of the amplified material is used for hybridization to the
microarray and, as such, the technique is also archival. Despite these advantages, adoption of
these approaches has been slower than for T7/IVT. Much of the skepticism surrounding PCR
amplification for global gene expression stems from concerns that PCR shows GC-content
biases, produces double-stranded products and is non-linear in nature [15]. Evolutions of the
PCR-based approach have addressed many of these concerns. Some groups have opted to
combine both PCR and T7/IVT approaches, which helps balance out some of the problems
of both techniques [37-39].
Another approach to amplification is a proprietary technique call RiboSPIA
from
NuGen Technologies. This technique utilizes a chimeric RNA/DNA primer which in the
presence of RNase H will displace the previous cDNA strand in order to create another
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