用于下游分析的环状RNA的反义Oligo Pulldown

Debojyoti Das; Aniruddha Das; Amaresh C. Panda

doi:10.21769/BioProtoc.4088

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Peer-reviewed

Antisense Oligo Pulldown of Circular RNA for Downstream Analysis

用于下游分析的环状RNA的反义Oligo Pulldown

DD Debojyoti Das ^*

AD Aniruddha Das ^*

Amaresh C. Panda email

(*contributed equally to this work) 发布: 2021年07月20日第11卷第14期 DOI: 10.21769/BioProtoc.4088 浏览次数: 4058

评审: Florian KarrethGopal PandiAnonymous reviewer(s)

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Cover of Nucleic Acids Research, featuring study using the protocol.

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Abstract

Circular RNAs (circRNAs) are a large family of noncoding RNA molecules that have emerged as novel regulators of gene expression by sequestering microRNAs (miRNAs) and RNA-binding proteins (RBPs). Several computational tools have been developed to predict circRNA interaction with target miRNAs and RBPs with a view to studying their potential effect on downstream target genes and cellular physiology. Biochemical assays, including reporter assays, AGO2 pulldown, ribonucleoprotein pulldown, and biotin-labeled RNA pulldown, are used to capture the association of miRNAs and RBPs with circRNAs. Only a few studies have used circRNA pulldown assays to capture the associated miRNAs and RBPs under physiological conditions. In this detailed protocol, the circRNA of interest (e.g., circHipk2) was captured using a biotin-labeled antisense oligo (ASO) targeting the circHipk2 backsplice junction sequence followed by pulldown with streptavidin-conjugated magnetic beads. The specific enrichment of circRNA was analyzed using reverse transcription quantitative PCR (RT-qPCR). Furthermore, the ASO pulldown assay can be coupled to miRNA RT-qPCR and western blotting analysis to confirm the association of miRNAs and RBPs predicted to interact with the target circRNA. In summary, the specific pulldown of circRNA using this quick and easy method makes it a useful tool for identifying and validating circRNA interaction with specific miRNAs and RBPs.

Keywords: CircRNAs (环状rna)

CircRNA pulldown (环状rna pulldown)

Antisense oligo (反义寡核苷酸)

Biotin pulldown (生物素pulldown)

RT-qPCR (RT-qPCR)

miRNA (miRNA)

RNA-binding proteins (rna结合蛋白)

Background

The RNA family can be broadly classified into coding (mRNAs) and noncoding RNAs. Interestingly, the vast majority of cellular RNAs are noncoding RNAs, including rRNA, lincRNA, miRNA, snRNA, snoRNA, tRNA, piRNA, and poorly characterized circular (circ) RNAs (Palazzo and Lee, 2015). The use of next-generation RNA sequencing and bioinformatics tools has uncovered more than a hundred thousand circRNAs in humans (Vromman et al., 2020). CircRNAs are generated from pre-mRNAs by head-to-tail splicing of specific exons by a process called backsplicing, which is regulated by transcription speed, RBPs, and inverted repeat sequences. Their length can vary from less than 100 nucleotides to a few thousand nucleotides. Depending on the sequence of circRNA origin from the parent gene, circRNAs have been classified into exonic circular RNA (circRNA), lariat-derived circular intronic RNA (ci-RNA), stable intronic sequence RNA (sisRNA), and exonic-intronic circular RNA (EIcircRNA) (Guria et al., 2019). Although a vast number of circRNAs have been identified in different cellular systems and disease models, only a fraction of circRNAs has been functionally characterized (Vromman et al., 2020). Recent evidence suggests that circRNAs play a critical role in regulating cellular events by interacting with miRNAs and RBPs (Guria et al., 2019). The majority of studies use computational tools to predict the association of circRNAs with cellular miRNAs and proteins that could regulate the expression of downstream target genes (Dudekula et al., 2016). Several biochemical techniques are currently used to analyze the circRNA-miRNA interaction, including luciferase reporter assays, AGO2 pulldown, and fluorescence in situ hybridization (FISH). Similarly, ribonucleoprotein pulldown assays using an antibody against the predicted RBP and biotin-labeled RNA pulldown assays to capture the target RBPs are used to validate the interaction of circRNA with specific RBPs. However, some of these methods are indirect assays to conclude whether the circRNA is associated with target miRNAs or RBPs. Our previous studies have successfully used circRNA pulldown assays with biotin-labeled antisense oligos targeting the circRNA backsplice junction sequence. Furthermore, circRNA-pulldown followed by RT-qPCR and western blotting analysis identified the miRNA or RBP associated with the circRNA of interest (Abdelmohsen et al., 2017; Panda et al., 2017; Pandey et al., 2020). Here, we adapted the published method to successfully pull down circHipk2 in βTC6 cells. The detailed protocol for the pulldown of circRNA to analyze the associated miRNA or RBP is described here.

Materials and Reagents

Nuclease-free 1.5-ml microcentrifuge tubes (Tarson, catalog number: 500010)
Nuclease-free 2-ml microcentrifuge tubes (Tarson, catalog number: 500020)
10 µl, 20 µl, 200 µl, and 1 ml tips (Tarson, catalog numbers: 524053, 528101, 528104, 528106)
96-well PCR plates (Thermo Fisher Scientific, catalog number: 4483485)
Nuclease-free water (Thermo Fisher Scientific, catalog number: 10977023)
0.25% Trypsin (Thermo Fisher Scientific, catalog number: 25200056)
Phosphate-buffered saline (PBS) (Sigma, catalog number: P4417-50TAB)
1 M Tris-HCl, pH 7.5 (Thermo Fisher Scientific, catalog number: 15567027)
1 M Tris-HCl, pH 8.0 (Thermo Fisher Scientific, catalog number: 15568025)
2 M Potassium chloride (Thermo Fisher Scientific, catalog number: AM9640G)
1 M Magnesium chloride (Thermo Fisher Scientific, catalog number: AM9530G)
5 M Sodium chloride (Thermo Fisher Scientific, catalog number: AM9759)
Nonidet P-40 (Amresco, catalog number: M158-100ML)
0.5 M EDTA, pH 8.0 (Amresco, catalog number: E177-100ML)
Triton X-100 (SRL, catalog number: 64518)
Murine RNase inhibitor (NEB, catalog number: M0314S, 40 U/µl)
20× Protease inhibitor (Sigma, catalog number: S8830)
Streptavidin Dynabeads (NEB, catalog number: S1420S)
TRIzol (Thermo Fisher Scientific, catalog number: 15596018)
Ethanol (HiMedia, catalog number: MB228)
Chloroform (SRL, catalog number: 96764)
Isopropanol (SRL, catalog number: 38445)
GlycoBlue^TM Coprecipitant (15 mg/ml) (Thermo Fisher Scientific, catalog number: AM9515)
Maxima reverse transcriptase (Thermo Fisher Scientific, catalog number: EP0743)
dNTP set, 100 mM solutions (Thermo Fisher Scientific, catalog number: R0181)
Random primers (Thermo Fisher Scientific, catalog number: 48190011)
PowerUp SYBR Green master mix (Thermo Fisher Scientific, catalog number: A25778)
MicroAmp optical adhesive film (Thermo Fisher Scientific, catalog number: 4311971)
Divergent DNA oligo primers synthesized by SIGMA for PCR amplification of circHipk2 (circHipk2-F: 5’-TCCAGACAACCGTACCGAGT-3’ and circHipk2-R: 5’-GGCACTTGATTGAAGGGTGT-3’)
Custom antisense oligos labeled with biotin-TEG synthesized by SIGMA for control (Ctrl-ASO: 5’-TGCGTAACGAACGACGAATCGTCGCAGATC-3’[BtnTg]) and circHipk2 (circHipk2-ASO: 5’-CATGTGAGGCCATACCGGTAGTATCTGGAT-3’[BtnTg])
3× SDS loading dye (NEB, catalog number: B7703S)
Polysome extraction buffer (PEB) (see Recipes)
2× Tris, EDTA, NaCl, Triton (TENT) buffer (see Recipes)
1× TENT (see Recipes)

Equipment

Manual pipette set, 2 µl, 20 µl, 200 µl, and 1 ml (various manufacturers)
Vortex mixer (Tarson, catalog number: 3001)
Magnetic stand (Tarson, catalog number: S1509S)
Tube rotator (Tarson, catalog number: 3071)
Refrigerated centrifuge (Eppendorf, model: 5810R)
Benchtop microfuge (Tarson, catalog number: 1010)
Thermomixer (Eppendorf, catalog number: 5384000012)
PCR machine (various manufacturers)
QuantStudio 3 real-time PCR system (Thermo Fisher Scientific, catalog number: A28567)

Software

UCSC genome browser (https://genome.ucsc.edu/)
Primer3 webtool (https://bioinfo.ut.ee/primer3/)
GeneRunner (http://www.generunner.net/)
QuantStudio 3 and 5 system software
Circinteractome website (https://circinteractome.nia.nih.gov/)

Procedure

English

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如何引用

Das, D., Das, A. and Panda, A. C. (2021). Antisense Oligo Pulldown of Circular RNA for Downstream Analysis. Bio-protocol 11(14): e4088. DOI: 10.21769/BioProtoc.4088.