What type of study is this?

This is a In Vitro Study study.

September 10, 2025

Design and in vitro transcription of shRNA with theNEB HiScribe™ T7 Quick High Yield RNA Synthesis Kit v1

CRChristian CR RenickeJohns Hopkins University PCPhillip A. ClevesJohns Hopkins University NSNatalie Swinhoe

Key Points

This protocol produces shRNA yields between 30 and 150 µg from a dsDNA template.
Using T7 RNA polymerase, this method is effective for RNAi applications in manipulating gene expression.
The shRNA design targets different mRNAs after electroporation into Aiptasia zygotes.
This in vitro transcription method is a reliable approach for generating shRNA for gene knockdown studies.

Abstract

This a protocol for design and in vitro transcription (IVT) of shRNA with T7 RNA polymerase from a dsDNA template created by annealing two complementary ssDNA oligos. A single reaction should produce between 30 and 150 µg shRNA depending on the sequence directly downstream of the T7 promoter (and to some part probably the overall sequence). The protocol was used to produce shRNA to target different mRNAs for RNAi-based knockdown after electroporation into Aiptasia zygotes.

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