Biomedical Engineering Reference
In-Depth Information
5.2 Methods and approaches
While several methods for amplification exist, we have opted to focus on two different
approaches: T7-based amplification using a commercial kit (MessageAmp
from Ambion;
Protocols 5.1-5.7) and our own in-house derived global-RT-PCR methodology [34];
Protocols 5.8-5.13).
™
5.2.1 T7 RNA polymerase-based
in vitro
transcription
While it is quite possible to put together a 'home-brew' version of the T7-amplification kits
following the numerous publications available, we have chosen a commercial amplification
kit that we have had good success with as an example for this chapter. Commercial kits
have the advantage of overall quality control of the components as well as technical support
for troubleshooting. As such, many researchers prefer to use a commercial kit for complex
protocols such as this. The Ambion MessageAmp
kit has been extensively used in the
literature. Here we present only a single round amplification procedure. It has been our
experience that if lower amounts of RNA need to be used, the global-RT-PCR procedure is
more reliable than a two-round T7-amplification.
™
PROTOCOL 5.1 First-Strand cDNA Synthesis Using MessageAmp
™
Kit (Ambion)
Equipment and reagents
•
T7 Oligo(dT) primer (MessageAmp
™
kit component, Ambion)
•
Reverse transcriptase (MessageAmp
™
kit component, Ambion)
•
RNase inhibitor (MessageAmp
™
kit component, Ambion)
•
10
×
first-strand buffer (MessageAmp
™
kit component, Ambion)
•
dNTP mix (MessageAmp
™
kit component, Ambion)
•
Nuclease-free water (Sigma)
•
Thermal cycler set at 70
◦
C
•
Hybridization oven or air incubator set at 42
◦
C.
Method
1 In a 0.2 ml microcentrifuge tube, combine the following reagents:
•
100-1000 ng of total RNA
a
•
1
μ
l of T7 Oligo(dT) primer
•
nuclease-free water to a final volume of 12
μ
l.
2 Incubate the reaction mixture at 70
◦
C for 10 min in a thermal cycler. Centrifuge briefly
to collect the sample at the bottom of the tube and place on ice.
