Automated High-Throughput mRNA Selection from Eukaryotic Total RNA

An athermal approach to mRNA enrichment from total RNA using a self‐immolative thioester linked nucleic acids (TENA) is described. Oligo(thymine) (oT) TENA has a six‐atom spacing between bases which allowed TENA to selectively base‐pair with polyadenine RNA. As a result of the neutral backbone of TENA and the hydrophobicity of the octanethiol end group, oT TENA is water insoluble and efficiently pulled down 93±2 % of EGFP mRNA at a concentration of 10 ng μL−1. Self‐immolative degradation of TENA upon ambient temperature exposure to nucleophilic buffer components (Tris, DTT) allowed recovery of 55±27 ng of mRNA from 3.1 μg of total RNA, which was not statistically different from the amount recovered using Dynabeads® mRNA DIRECT Kit (89±24 ng). Gene expression as measured by RT‐qPCR was comparable for both enrichment methods, suggesting that the mild conditions required for enrichment of mRNA using oT TENA are compatible with RT‐qPCR and other downstream molecular biology applications.

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Section: Figurementioning

confidence: 99%

Athermal, Chemically Triggered Release of RNA from Thioester Nucleic Acids

et al. 2021

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“…These tasks are well performed by molecular biological workstations, which are capable to accommodate special equipment such as vacuum manifolds, magnetic separators, shakers, incubators or indexed centrifuges on, beneath or under the work deck. Examples for genomics specific tasks reported in the literature include identification and picking of colonies, 15 purification of DNA from large samples, 16 automated sample-preparation technologies in genome sequencing projects, 17 high throughput probe production for DNA microarrays, 18 plasmid purification, 19 high throughput DNA extraction methods for PCR, 20 automated cycle sequencing, 21 purification of BigDye Ter-minator fluorescent DNA sequencing reaction, 22 purification of PCR products, 23 automated agarose gel electrophoresis, 24 high throughput RNA purification, 25,26 and printing of microarrays using liquid-handling robots. 27 However, some tasks are difficult to automate at all, such as initial sample (e.g.…”

Section: Considerations For Choosing Automationsmentioning

confidence: 99%

Liquid-Handling Robotic Workstations for Functional Genomics

Lorenz

2004

JALA: Journal of the Association for Laboratory Automation

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More and more functional genomics laboratories are willing to invest in robotic workstations due to the higher throughput liquid-handling intensive nature of the work. In this report, the features of robotic workstations important for functional genomics are discussed. Workstations for functional genomics are useful for replication of clone sets, PCR and sequencing set-up and clean-up, hit picking, gel loading, and nucleic acid purification procedures. Workstations not only increase throughput, but also ensure that assay steps are performed consistently, human error- and learning curve-free from run to run. Workstations fit on a laboratory bench and may have several robotic arms in different configurations. Available configurations include grippers and single-, oligo-, or multi-probe heads, with uniform or independent spanning, and liquid-level detection or tracking options. Most hardware and software packages can be designed for integration with other automated devices, building towards a fully automated system.

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“…For eukaryotic samples, the most common isolation strategies rely on hybridization of oligo(thymine) (oT) with the poly(adenine) (poly(A)) tail of mRNA, which enables efficient separation from ribosomal and other RNAs from the sample. [9][10][11][12][13][14] Specifically, oT DNA conjugated to solid supports (i.e., column packing material or magnetic beads) binds to the poly(A) tail and facilitates the specific sequestration of mRNA. Such techniques require the use of expensive kits or columns that require specialized equipment and refrigerated storage, which might not be accessible in certain situations.…”

mentioning

confidence: 99%

Athermal, Chemically Triggered Release of RNA from Thioester Nucleic Acids

et al. 2021

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An athermal approach to mRNA enrichment from total RNA using a self-immolative thioester linked nucleic acids (TENA) is described. Oligo(thymine) (oT) TENA has a six-atom spacing between bases which allowed TENA to selectively base-pair with polyadenine RNA. As a result of the neutral backbone of TENA and the hydrophobicity of the octanethiol end group, oT TENA is water insoluble and efficiently pulled down 93 AE 2 % of EGFP mRNA at a concentration of 10 ng mL À1 . Self-immolative degradation of TENA upon ambient temperature exposure to nucleophilic buffer components (Tris, DTT) allowed recovery of 55 AE 27 ng of mRNA from 3.1 mg of total RNA, which was not statistically different from the amount recovered using Dynabeads mRNA DIRECT Kit (89 AE 24 ng). Gene expression as measured by RT-qPCR was comparable for both enrichment methods, suggesting that the mild conditions required for enrichment of mRNA using oT TENA are compatible with RT-qPCR and other downstream molecular biology applications.

show abstract

Automated High-Throughput mRNA Selection from Eukaryotic Total RNA

Cited by 3 publications

References 5 publications

Athermal, Chemically Triggered Release of RNA from Thioester Nucleic Acids

Athermal, Chemically Triggered Release of RNA from Thioester Nucleic Acids

Liquid-Handling Robotic Workstations for Functional Genomics

Athermal, Chemically Triggered Release of RNA from Thioester Nucleic Acids

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