| 1. |
- Baronti, L, et al.
(author)
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A guide to large-scale RNA sample preparation
- 2018
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In: Analytical and bioanalytical chemistry. - : Springer Science and Business Media LLC. - 1618-2650 .- 1618-2642. ; 410:14, s. 3239-3252
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Journal article (peer-reviewed)
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| 2. |
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| 3. |
- Feyrer, H, et al.
(author)
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One-Pot Production of RNA in High Yield and Purity Through Cleaving Tandem Transcripts
- 2020
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In: Molecules (Basel, Switzerland). - : MDPI AG. - 1420-3049. ; 25:5
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Journal article (peer-reviewed)abstract
- There is an increasing demand for efficient and robust production of short RNA molecules in both pharmaceutics and research. A standard method is in vitro transcription by T7 RNA polymerase. This method is sequence-dependent on efficiency and is limited to products longer than ~12 nucleotides. Additionally, the native initiation sequence is required to achieve high yields, putting a strain on sequence variability. Deviations from this sequence can lead to side products, requiring laborious purification, further decreasing yield. We here present transcribing tandem repeats of the target RNA sequence followed by site-specific cleavage to obtain RNA in high purity and yield. This approach makes use of a plasmid DNA template and RNase H-directed cleavage of the transcript. The method is simpler and faster than previous protocols, as it can be performed as one pot synthesis and provides at the same time higher yields of RNA.
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| 4. |
- Karlsson, H, et al.
(author)
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A robust and versatile method for production and purification of large-scale RNA samples for structural biology
- 2020
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In: RNA (New York, N.Y.). - : Cold Spring Harbor Laboratory. - 1469-9001 .- 1355-8382. ; 26:8, s. 1023-1037
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Journal article (peer-reviewed)abstract
- Recent findings in genome-wide transcriptomics revealed that RNAs are involved in almost every biological process, across all domains of life. The characterization of native RNAs of unknown function and structure is particularly challenging due to their typical low abundance in the cell and the inherent sensitivity toward ubiquitous RNA degrading enzymes. Therefore, robust in vitro synthesis and extensive work-up methods are often needed to obtain samples amenable for biochemical, biophysical, and structural studies. Here, we present a protocol that combines the most recent advances in T7 in vitro transcription methodology with reverse phase ion pairing and ion exchange HPLC purification of RNAs for the production of yield-optimized large-scale samples. The method is easy to follow, robust and suitable for users with little or no experience within the field of biochemistry or chromatography. The complete execution of this method, for example, for production of isotopically labeled NMR samples, can be performed in less than a week.
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