Name: biotin-based pulldown assay to validate mrna targets of cellular mirnas
Uploaded: 2018-06-12T15:00:00
Description: Watch this Scientific Journal Video about Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs at JoVE.com

This work was partly supported by National Institutes of Health (NIH) Grants DA037779 (to J.P.), DA024558, DA30896, DA033892, DA021471, AI22960 and MD007586 (to C.D.). The work was also supported by the RCMI Grant G12MD007586, the Vanderbilt CTSA Grant UL1RR024975, the Meharry Translational Research Center (MeTRC) CTSA grant (U54 RR026140 from NCRR/NIH, the U54 Grant MD007593 from NIMHD/NIH, and the Tennessee Center for AIDS Research (P30 AI110527).

1. Transfection of 3&#39;-biotinylated miRNA
NOTE: Perform the following steps inside a sterile laminar flow hood.
<ol>
	<li>Seed 4 x 105&#8211;5 x 105 HEK-293T cells per well in 2 mL of complete Dulbecco&#39;s Modified Eagle Medium (DMEM) [DMEM supplemented with 10% Fetal Bovine serum (FBS) and 1x Pen-strep antibiotic]. Culture the cells in an incubator set at 37 &#176;C, 5% CO2 overnight.</li>
	<li>The next day, check the health and adherence of the plated ...

Identification of mRNA targets of cellular miRNAs is important for understanding their regulatory function. Several computational tools are employed to predict targets based on seed sequence complementarity and conservation of target sequences19,20. Although these tools are valuable, they can generate high levels of both false positives and false negatives. Therefore, these predictions are coupled with experimental methods such as immunoprecipitation of RISC comp...

MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate protein expression1. Precursors of miRNA reside in clusters through many regions of the genome, most frequently within the intergenic regions and introns of protein-coding genes2,3. Biogenesis of miRNAs involve transcription of the pri-miRNAs from miRNA-encoding genes4. The pri-miRNAs undergo sequential processing, first in the nucleus and then in the cytoplasm to generate single stranded mature miRNAs4,5. Subsequently, the mature miRNAs are incorporated into the RNA-induced silencing complex (RISC): a multimeric protein-RNA complex that includes a member of the Argonaute family of proteins for target recognition4,5,6,7. Mature miRNAs in the RISC complex predominantly bind to the 3&#39; UTR of target mRNAs8,9,10 but can also bind occasionally in the coding and the 5&#39;UTR region of the mRNA10,11. The binding of miRNA to the mRNA sequences results in translational silencing12,13,14 and in some cases mRNA destabilization15. Since a single miRNA can target many mRNAs, these regulatory molecules are involved in almost every cellular process and have been implicated in various disease conditions16,17,18.
Detailed understanding of how miRNAs regulate cellular pathways requires identification of the target mRNAs. Multiple bioinformatics platforms are available to predict putative miRNA:mRNA interactions19,20. These predictions rely on perfect Watson-Crick base pairing between the 2&#8211;8 nucleotide seed sequence of the miRNA and a complementary sequence within the target mRNA4,21. Additionally, these tools generate secondary structure of the miRNA:mRNA duplex, calculate thermodynamic parameters of this molecular interaction and show conservation of the binding sites across species to enhance functional relevance of the target prediction. Unfortunately, these tools also have the limitation of predicting false positive targets at a very high rate (~27&#8211;70%)15,22. Most importantly, these in silico platforms fail to recognize the non-canonical interactions of miRNAs with their targets23. Therefore, such predictive analyses are often combined with experimental methods to validate functionally relevant targets.
Multiple approaches have been developed to experimentally validate miRNA:mRNA interaction. Genetic experiments using miRNA mimics, sponges and inhibitors that alter the levels of miRNAs in the cell provide clues for its regulatory effect on the target gene expression24,25,26. Additionally, reporter based assays via co-transfection of a clone containing the 3&#39; UTR region of the target mRNA and miRNA mimics or inhibitors into cells provide evidence of the regulatory function of miRNAs26. While these methods are crucial to study post-transcriptional regulation of gene expression by miRNAs, transfection efficiency and pleotropic effects of alterations in cellular miRNA levels are major limitations of these genetic approaches23,26. Therefore, complementary biochemical methods that probe direct interaction between miRNA and its target are employed to better understand the cellular function of miRNAs.
One widely-used method to study direct interaction between miRNA and its target is immunoprecipitation of the RISC complex followed by the detection of mRNA target within the complex27,28,29,30. Recently, an improved RISC trap method was also utilized to identify miRNA targets; it couples stabilization of targets within the RISC-miRNA-mRNA intermediates with the purification of mRNA targets. However, these methodsface the inherent challenges of non-specific interactions between RNA and RNA-binding proteins that are usually segregated by cellular compartments31,32. In addition, these assays are dependent on the presence of AGO2 protein for immunoprecipitation of RISC complex33. Given that AGO2 is not the only argonaute to mediate efficient miRNA:mRNA interactions, exclusion of other argonautes could lead to biased results34. Therefore, alternative strategies are needed to study direct binding of miRNA to mRNA.
In this report, we elaborate a one-step approach for probing direct interaction between a miRNA and its target mRNA. First, 3&#39; biotinylated locked nucleic acid (LNA) miRNA mimics are transfected into mammalian cells. Then, the miRNA:mRNA complex in the cellular lysate is captured using streptavidin coated magnetic beads. The mRNA target bound to its complementary miRNA is quantified using qPCR.

<table>
	<tbody>
		<tr>
			<td>Cell line- HEK293T cells</td>
			<td>ATCC</td>
			<td>CRL-3216</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat-inactivated Fetal bovine serum (Hi-FBS)</td>
			<td>GIBCO/Thermofisher</td>
			<td>10438-026</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td>GIBCO/Thermofisher</td>
			<td>11995-065</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS) (1x)</td>
			<td>GIBCO/Thermofisher</td>
			<td>20012-027</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)&#160;</td>
			<td>GIBCO/Thermofisher</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin solution (100x)</td>
			<td>Cellgro/Mediatech</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase&#160;</td>
			<td>Ambion</td>
			<td>AM2238</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM Reduced serum media</td>
			<td>GIBCO/Thermofisher</td>
			<td>3198-088</td>
			<td></td>
		</tr>
		<tr>
			<td>Liopfectamine 2000 Transfection reagent</td>
			<td>Invitrogen/ Thermofisher</td>
			<td>11668-019</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated hsa-miR-125b</td>
			<td>Exiqon</td>
			<td>339178/ID-27281622</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated scrambled control</td>
			<td>Exiqon</td>
			<td>479997-671/ID-714884</td>
			<td></td>
		</tr>
		<tr>
			<td>IGEPAL</td>
			<td>Sigma-Aldrich</td>
			<td>18896</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin Magnetic beads</td>
			<td>Pierce</td>
			<td>88816</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast tRNA</td>
			<td>Invitrogen/Thermofisher</td>
			<td>15401-011</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl) solution</td>
			<td>Sigma-Aldrich</td>
			<td>60142</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride (MgCl2) solution</td>
			<td>Sigma-Aldrich</td>
			<td>M1028</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Sigma-Aldrich</td>
			<td>795429</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediamine tetraacetic acid, disodium salt (EDTA) solution, 0.5 M, pH 8.0</td>
			<td>Sigma-Aldrich</td>
			<td>E7889</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Sigma-Aldrich</td>
			<td>43815</td>
			<td></td>
		</tr>
		<tr>
			<td>Superase</td>
			<td>Ambion/Thermofisher</td>
			<td>AM2694</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>P8340</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse/DNAse free water</td>
			<td>GIBCO/Thermofisher</td>
			<td>10977-015</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy extraction kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>iScript-Select cDNA synthesis kit</td>
			<td>Biorad</td>
			<td>170-8897</td>
			<td></td>
		</tr>
		<tr>
			<td>qPCR Primers&#160;</td>
			<td>Invitrogen/Thermofisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>iTaq-Universal SYBR green supermix</td>
			<td>Biorad</td>
			<td>172-5120</td>
			<td></td>
		</tr>
		<tr>
			<td>DynaMag-Spin Magnet</td>
			<td>Invitrogen/Thermofisher</td>
			<td>12320D</td>
			<td></td>
		</tr>
	</tbody>
</table>

biotin-based pulldown assay to validate mrna targets of cellular mirnas

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; untranslated region (UTR) of target mRNA to inhibit protein translation or in some instances cause mRNA degradation. The binding of the miRNA to the 3&#39; UTR of the target mRNA is mediated by a 2&#8211;8 nucleotide seed sequence at the 5&#39; end of miRNA. While the role of miRNAs as cellular regulatory molecules is well established, identification of the target mRNAs with functional relevance remains a challenge. Bioinformatic tools have been employed to predict sequences within the 3&#39; UTR of mRNAs as potential targets for miRNA binding. These tools have also been utilized to determine the evolutionary conservation of such sequences among related species in an attempt to predict functional role. However, these computational methods often generate false positive results and are limited to predicting canonical interaction between miRNA and mRNA. Therefore, experimental procedures that measure direct binding of miRNA to its mRNA target are necessary to establish functional interaction. In this report, we describe a sensitive method for validating direct interaction between the cellular miRNA miR-125b and the 3&#39; UTR of PARP-1 mRNA. We elaborate a protocol in which synthetic biotinylated-miRNA mimics were transfected into mammalian cells and the miRNA-mRNA complex in the cellular lysate was pulled down with streptavidin-coated magnetic beads. Finally, the target mRNA in the pulled-down nucleic acid complex was quantified using a qPCR-based strategy.

MiRNAs regulate cellular processes by binding to target mRNAs. Therefore, identifying mRNA targets are a key to understand miRNA function. Here we elaborate a method for identifying mRNA target of cellular miRNA. This protocol is adapted from Wani et al.35 with the modification of utilizing biotinylated LNA-based miRNA mimics to pulldown target mRNA. The use of LNA-based oligonucleotide mimics enhances specificity of target binding owing to the higher stab...

Watch this Scientific Journal Video about Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs at JoVE.com

Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs

This report describes a fast and reliable method for validating mRNA targets of cellular miRNAs. The method uses synthetic biotinylated Locked Nucleic Acid (LNA)-based miRNA mimics to capture target mRNA. Subsequently, streptavidin-coated magnetic beads are employed to pulldown the target mRNA for quantification by qPCR polymerase chain reaction.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; ...

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