Title

High Yield Extraction of Clean RNA from Variovorax paradoxus

Poster Number

14B

Lead Author Major

Pre-Dentistry

Lead Author Status

Sophomore

Second Author Major

Pre-Dentistry

Second Author Status

Sophomore

Third Author Major

Music Therapy

Third Author Status

Junior

Fourth Author Major

Pre-Dentistry

Fourth Author Status

Senior

Format

Poster Presentation (Research Day, April 30)

Faculty Mentor Name

Paul Orwin

Faculty Mentor Department

Biological Sciences

Abstract/Artist Statement

Variovorax paradoxus is a Gram-negative soil proteobacterium that produces biofilms on plant roots and plays a major role in the rhizosphere of Arabidopsis thaliana. Our research group is examining the mechanisms of biofilm formation in this organism with a focus on the role of Toxin-Antitoxin (TA) systems. As part of this work, we need to be able to purify RNA from different growth phases to be able to use for transcriptome analysis. This will allow us to identify patterns of gene expression associated with the biofilm lifestyle.

The work reported here is focused on gathering samples of purified RNA from different V. paradoxus strains exponential-phase growth. This RNA will be compared to samples derived from biofilms to identify specific differentially expressed loci. Highly purified RNA is necessary for RNA-seq, the most effective strategy for determining global patterns of gene expression. However, RNAse activity and contamination with other nucleic acids are significant obstacles. Here we describe a set of experiments to optimize RNA yield and purity in preparation for that future work.

We have developed a strategy for culture growth and RNA extraction that reliably yields large amounts of high-quality RNA (OD260/280 ratio ~2.0) using a Zymo RNA purification kit. In addition, we present growth data on selected strains in batch culture to facilitate the process of reliably extracting RNA from bacteria in the exponential phase. The purified RNA will be sequenced and compared with biofilm growth to identify TA systems that are differentially regulated.

Location

Information Commons, William Knox Holt Memorial Library and Learning Center

Start Date

30-4-2022 10:00 AM

End Date

30-4-2022 12:00 PM

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Apr 30th, 10:00 AM Apr 30th, 12:00 PM

High Yield Extraction of Clean RNA from Variovorax paradoxus

Information Commons, William Knox Holt Memorial Library and Learning Center

Variovorax paradoxus is a Gram-negative soil proteobacterium that produces biofilms on plant roots and plays a major role in the rhizosphere of Arabidopsis thaliana. Our research group is examining the mechanisms of biofilm formation in this organism with a focus on the role of Toxin-Antitoxin (TA) systems. As part of this work, we need to be able to purify RNA from different growth phases to be able to use for transcriptome analysis. This will allow us to identify patterns of gene expression associated with the biofilm lifestyle.

The work reported here is focused on gathering samples of purified RNA from different V. paradoxus strains exponential-phase growth. This RNA will be compared to samples derived from biofilms to identify specific differentially expressed loci. Highly purified RNA is necessary for RNA-seq, the most effective strategy for determining global patterns of gene expression. However, RNAse activity and contamination with other nucleic acids are significant obstacles. Here we describe a set of experiments to optimize RNA yield and purity in preparation for that future work.

We have developed a strategy for culture growth and RNA extraction that reliably yields large amounts of high-quality RNA (OD260/280 ratio ~2.0) using a Zymo RNA purification kit. In addition, we present growth data on selected strains in batch culture to facilitate the process of reliably extracting RNA from bacteria in the exponential phase. The purified RNA will be sequenced and compared with biofilm growth to identify TA systems that are differentially regulated.