I ran an in vitro transcription reaction (IVT) from a 4.8kb plasmid, with the expected product of 2.1kb. After 3 hours of reaction time, I saw two bands of approximately 1.5 kb in length on an agarose TBE gel. I wonder why I am getting shorter RNAs than expected.
I am not an expert in this field, but I am very interested and have researched to find an answer. I received some assistance from tlooto.com for this response. Could you please review the response below to see if it is correct?
The presence of shorter RNA bands of approximately 1.5 kb instead of the expected 2.1 kb product in your in vitro transcription (IVT) reaction could be due to several factors. Premature termination of transcription may occur because of secondary structures in your RNA template, which can hinder polymerase progress [1]. Additionally, RNA degradation might be a factor, possibly due to RNase contamination, so ensure all reagents and equipment used are RNase-free [3]. Verify the integrity and sequence of your plasmid to confirm the correct positioning of the transcription start site. Additionally, optimize reaction conditions like Mg2+ concentration and temperature to improve transcription efficiency [2]. Consider adjusting these parameters to achieve the desired full-length transcript.
Reference
[1]
Rigby, C. V., Sabsay, K. R., Bisht, K., Eggink, D., Jalal, H., & Velthuis, A. J. T. t. (2023). Evolution of transient RNA structure–RNA polymerase interactions in respiratory RNA virus genomes. Virus Evolution, 9.
[2]
Mu, X., & Hur, S. (2021). Immunogenicity of In Vitro-Transcribed RNA.. Accounts of Chemical Research.
[3]
Morton, D. J., Kuiper, E., Jones, S. K., Leung, S. W., Corbett, A., & Fasken, M. B. (2018). The RNA exosome and RNA exosome-linked disease. RNA, 24, 127 - 142.
To solve problems with IVT you could use the article posted by Kelly Grooms “In Vitro Transcription: Common Causes of Reaction Failure” on the website https://www.promegaconnections.com/in-vitro-transcription-common-causes-of-reaction-failure/.
Since the exact sequence isn’t known, I can’t say for certain, but there might be a terminator sequence in the sequence. Even if it’s not in a perfect form, it could still show a weak termination efficiency. It might be helpful to check if the sequence at that specific length corresponds to the termination sequence of the phage used.
Is it on linearized plasmid? blunt ?5' or 3' overhang? or on a plasmid with a T7 terminator sequence? polyU (poly(T) on your DNA sequence; at least four in a raw) is a termination signal for T7 ....
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