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Old 12-01-2011, 07:21 AM   #14
TonyBrooks
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Location: London

Join Date: Jun 2009
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T7 IVT is not a library prep method. It is a way of amplifying poly A+ RNA. It produces single stranded cRNA (although it does keep it's strandedness as it's only amplified from one strand). It's mostly used in microarrays where the input needs to be single stranded material.

The "dUTP" method is a library prep method. The only amplification is at the end of the protocol when the sample is enriched for adapter ligated material by PCR. Before this step, the second strand (marked with uracil) is degraded to only the first strand is enriched by PCR.

Amplification is a red herring here. You can't really compare dUTP and T7 IVT as they are predominantly used for different technologies. Although both methods retain strandedness, it's not an issue with T7 IVT as the microarray will only have probes where the strand of the mRNA is known. Strandedness is much more important in sequencing as you want to know which strand a read has come from to identify novel transcripts.
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