Biomedical Engineering Reference
In-Depth Information
10 Centrifuge each column at 13 800
g
for 1 min to elute the purified labeled cDNA.
ee
11 Evaporate each cDNA sample to dryness using a vacuum centrifugation on high-heat
setting, taking care not to dry the sample excessively.
ff
Notes
ee
If you wish to stop the protocol at any point and resume the next day you can do so after this
purification step. Simply freeze the fluor-labeled double-stranded cDNA at
−
20
◦
C. The material
is stable for several days.
ff
This allows you to bring the sample up in whatever volume is appropriate for your hybridization
step based on the platform you are using.
5.3 Troubleshooting
•
Underrepresentation of RNA species by T7-amplification method
: Owing to the higher
order secondary and tertiary structure of many mRNA species, it is not uncommon for
some RNAs to be underrepresented in the T7-amplification method. This is not necessarily
a problem that is unique to amplification; however, the effect tends to be magnified under
such conditions. The effect is most prominent in the initial cDNA production step (during
RT [42]). It is possible to mitigate the effects of secondary structures in RNA by using
SuperScript III rather than SuperScript II and performing the RT reaction at 50
◦
Cfor
more robust cDNA production.
•
Underrepresentation of RNA species by global-RT-PCR method
: The global-RT-PCR
method uses SuperScript III at 50
◦
C for the RT step and, as such, the underrepresenta-
tion of certain RNAs is generally not attributable to higher order secondary and tertiary
structures. In the global-RT-PCR reaction, the ability to control the amplimer length leads
to a highly robust and reproducible technique; however, this does lead to an extreme 3
bias which may not be compatible with all array types (probes which are much beyond
300 bp from the polyA tail will be less reliable). It is possible to substitute random primers
rather than oligo-dT to provide better coverage of the entire gene, but we have not yet
optimized such a procedure. Another option is to use arrays with extreme 3
-bias, such as
the X3P arrays from Affymetrix (this would require altering the protocol to incorporate
biotin rather than fluorescent moieties). This protocol also works well with cDNA arrays,
which are less common now, but are by nature almost always inclusive of the 3
end of
the gene.
•
Low yield from T7-amplification method
: The RNA being produced during the IVT
reaction may not be very stable at the optimal temperature for the T7 polymerase. It has
been shown that significant RNA degradation can be detected after 4 h of IVT [43]. Inter-
estingly, the impact of this finding has not been fully realized, as most of the commercial
kits (including those for the Affymetrix and Agilent platforms) still recommend relatively
long (6 h to overnight) incubations during IVT. If, however, it appears that RNA quality
is a problem or that longer reactions are actually providing a diminished yield of aRNA,
then it may be beneficial to try shorter IVT reactions of less than 4 h. The result will be
decreased sensitivity, which may require additional rounds of T7-based amplification.
