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Generation of Amplified Sense RNA by T7 IVT
To generate labeled hybridization targets
for microRNA microarrays, the amplified cDNA can be used as template for
T7-based In Vitro transcription (T7 IVT) in the presence of fluorescent
labeled nucleotides by standard methods. An example of a T7 IVT experiment
is shown in Fig. 4A and B.
Figure 4A. 50 ng of a pool of RNA from
4 different tissues was
amplified with the SBI kit and purified. A small sample of the amplified
cDNA was subjected to T7 IVT and then electrophoresed on a 3% agarose gel.
The yield of amplified RNA was 22 μg.
Figure 4B. A small sample of the
amplified sense RNA from Figure 4A was used as a template for T7 IVT. The
amplified sense RNAs were then tested for the presence of 5 small non-coding
RNAs, including 3 microRNAs (181a, 24, and 150) by RT-PCR. All 5 small RNAs
were present in the amplified RNA.
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