Dataset Information

A universal and cost-effective method for general and specific RNA-protein complex isolation_RBPome

ABSTRACT: The establishment of a protocol isolating specific RNPs by combining a total RNP isolation with an antisense probe-mediated specific RNP isolation

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Leaf, Diploid Cell

SUBMITTER: Roosje Van Ende

LAB HEAD: Koen Geuten

PROVIDER: PXD031578 | Pride | 2023-05-10

REPOSITORIES: Pride

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Publications

A widely applicable and cost-effective method for specific RNA-protein complex isolation.

Balzarini Sam S Van Ende Roosje R Voet Arnout A Geuten Koen K

Scientific reports 20230427 1

Although methodological advances have been made over the past years, a widely applicable, easily scalable and cost-effective procedure that can be routinely used to isolate specific ribonucleoprotein complexes (RNPs) remains elusive. We describe the "Silica-based Acidic Phase Separation (SAPS)-capture" workflow. This versatile method combines previously described techniques in a cost-effective, optimal and widely applicable protocol. The specific RNP isolation procedure is performed on a pre-pur ...[more]

PMID: 37106019

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Project description:Circadian behaviors are regulated by intrinsic biological clocks consisting of central molecular oscillators and output pathways. Despite significant progress in elucidating the central timekeeping mechanisms, the molecular pathways coupling the circadian pacemaker to overt rhythmic behavior and physiology remain elusive. The Drosophila LARK RNA-binding protein is a candidate for such a coupling factor. Previous research indicates that LARK functions downstream of the clock to mediate behavioral outputs. To better understand the roles of LARK in the Drosophila circadian system, we sought to identify RNA molecules associated with LARK in vivo, using a novel strategy that involves capturing the RNA ligands by immunoprecipitation, visualizing the captured RNAs using whole gene microarrays, and identifying functionally relevant targets through genetic screens. Experiment Overall Design: LARK-containing ribonucleoprotein complexes (LARK-RNPs) were precipitated from lysates of hand-dissected pharate adult brains using an affinity-purified anti-LARK antibody (around 1000 brains were used per immunoprecipitation experiment). A portion of each lysate was saved prior to immunoprecipitations (IPs) in order to measure the relative abundance of transcripts in a total RNA sample. RNAs extracted from the LARK-RNP and total RNA samples were labeled and hybridized to Drosophila whole-genome gene microarrays; signal intensities for individual genes were compared between samples to identify those RNAs that were enriched by immunoprecipitation (relative to their abundances in total RNA). RNAs that were selectively enriched in the LARK-RNP samples were considered to be potential targets of the RNA-binding protein. Experiment Overall Design: Due to the difficulty to dissect large amount of fly brains, only two such immunoprecipitation experiments were performed, each generating an IP RNA sample and a total RNA (control) sample. The amount of RNAs obtained from IP is very small thus only one array is used for each sample - i.e. there are only biological replicates and no technical replicate.

Dataset Information

A universal and cost-effective method for general and specific RNA-protein complex isolation_RBPome

Publications

A widely applicable and cost-effective method for specific RNA-protein complex isolation.

Similar Datasets

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