Name: highly efficient ligation of small rna molecules for microrna quantitation by high-throughput sequencing
Uploaded: 2014-11-18T14:00:00.000+00:00
Duration: 14 min 15 s
Description: Watch this Scientific Journal Video about Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing at JoVE.com

The authors would like to thank members of the Yi laboratory especially Zhaojie Zhang for fruitful discussions regarding linker design and ligation efficiencies, as well as the American Cancer Society for supporting this work through a postdoctoral fellowship (#125209) to J.E.L. Research reported in this publication was also supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number R01AR059697 (to R.Y.) and a research grant from the Linda Crnic Institute for Down Syndrome. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

NOTE: It is critical to maintain RNase-free conditions during the entire procedure.
1. Adenylation of 3&#8217; Linker
<ol>
	<li>Dilute DNA oligonucleotide designed for 3&#8217; ligation reactions to 100 &#956;M in nuclease-free H2O. 
	NOTE: The oligonucleotide should have a 5&#8217; phosphate group, a randomized dinucleotide at the 5&#8217; end, and a 3&#8217; dideoxycytosine. The 5&#8217; phosphate group is a requirement of the Mth enzyme for efficient 5&#8217; adenylation10. The phosphate can be added by pre-treatment of the oligonucleotide with a polynucleotide kinase, or ordered with the modification directly. The randomized dinucleotide at the 5&#8217; end is important for minimizing the sequence preference which RNA ligases exhibit for certain end-end joining reactions over others6. The 3&#8217; dideoxycytosine places a blocking element at the 3&#8217; end of the oligonucleotide to inhibit the formation of linker-linker concatamers during subsequent ligation steps and also serves to block the 3&#8217; end of the miRNA-Linker molecule formed at the end of the 3&#8217; ligation reaction11. The oligonucleotide linker sequence chosen for the unbiased miR-Seq method is as follows: 5&#8217;PO4NNTGGAATTCTCGGGTGCCAAGG-dideoxyC3&#8217;.</li>
	<li>Assemble the following adenylation reaction: 200 pmol 3&#8217; linker oligonucleotide, 4 &#956;l 10x&#160;5&#8217; DNA adenylation buffer, 4 &#956;l 1 mM ATP, 4 &#956;l Mth RNA ligase, nuclease-free H2O to a final volume of 40 &#956;l.</li>
	<li>Incubate reaction at 65 &#176;C for 1 hr. Terminate reaction by heating to 85 &#176;C for 5 min.</li>
	<li>Precipitate adenylated linker by the addition of 2.5 volumes of 100% ethanol and 1/3 volume of 10 M ammonium acetate to remove residual ATP and to prevent unanticipated ligations in future reactions.
	<ol>
		<li>Briefly, mix the alcohol, salt, and oligo solution to homogeneity, spinning at full speed in a microcentrifuge (~15,000 x g) at 4 &#176;C for 20 min. Wash the pellet in 70% ethanol, air dry, and resuspend in the appropriate volume of nuclease-free H2O to yield 50-100 &#956;M of adenylated oligo.</li>
	</ol>
	</li>
	<li>Confirm adenylation by running an 18% polyacrylamide gel.
	<ol>
		<li>Prepare the gel by mixing the following together: 6.3 g urea, 6.75 ml of 40% acrylamide, and 1.5 ml of 10x&#160;TBE. Add H2O to 15 ml.</li>
		<li>Mix until urea is completely dissolved and add 150 &#181;l 10% (w/v) ammonium persulfate (APS) and 15 &#181;l tetramethlyethylenediamine (TEMED).</li>
		<li>Once gel has set, pre-run for 30 min at 24 V/cm of gel width with 1x&#160;TBE running buffer at room temperature. Once 30 min is up flush wells with running buffer.</li>
		<li>Mix 5 pmol of adenylated oligonucleotides with and equal volume of 2x&#160;denaturing RNA loading buffer (95% (v/v) Formamide, 0.02% (w/v) SDS, 0.02% bromophenol blue (w/v),0.01% (w/v) Xylene Cyanol, 1.0 mM EDTA), heat briefly to 75 &#176;C, and snap cooled on ice. Dispense entire sample into the well of the gel. Also include an identical amount of non-adenylated control, and low molecular weight size standards.</li>
		<li>Run the gel at 24 V/cm until the bromophenol blue is &#190; of the way to the bottom of the gel. Disassemble apparatus, post-stain gel with 1x&#160;sybr-gold solution in 1x&#160;TBE and visualize on UV-transilluminator box (Figure 2). 
		NOTE: Gel purify the adenylated oligo in a manner similar to that described above if &#8220;no input RNA&#8221; controls are resulting in a high amount of PCR product as compared to samples with input RNA.</li>
	</ol>
	</li>
</ol>
2. Linker Ligation to 3&#8217; End of miRNA
<ol>
	<li>3&#8217; Ligation
	<ol>
		<li>Assemble the following components on ice in the order listed: 1.0 &#956;l 50% (w/v) PEG 8000, 0.5 &#956;l 10x&#160;RNA ligase buffer (500 mM Tris-HCl pH 7.5, 100 mM MgCl2, 10 mM DTT), 1.0 &#956;l adenylated 3&#8217; Linker (100 &#956;M), 0.5 &#956;l RNase inhibitor, 0.5-8.0 &#956;g total RNA, 0.5 &#956;l T4 RNA ligase 2, nuclease-free H2O to a final volume of 5 &#956;l.</li>
		<li>Mix the reaction by pipetting as the solution is too viscous to vortex due to the high concentration of PEG 8000. Once thoroughly mixed, place the reaction in a thermal cycler with a heated lid. Set the block to 16 &#176;C, and incubate for 4 hr. 
		NOTE: The reaction may be scaled up to a final volume of 20 &#956;l without any loss in ligation efficiency. React for 4 hr&#160;at ambient temperature or overnight at 4 &#176;C to yield essentially identical ligation efficiencies.</li>
	</ol>
	</li>
	<li>Gel Purification
	<ol>
		<li>Following 3&#8217; ligation, mix the reaction with an equal volume of 2x&#160;RNA loading buffer. Heat to 70 &#176;C for 5 min, and snap cool on ice.</li>
		<li>Run the sample on a 15% polyacrylamide gel for PAGE purification.
		<ol>
			<li>Prepare a 15% polyacrylamide gel containing 7 M urea, in a fashion similar to that described previously in step 1.5.</li>
			<li>Pre-run the gel at 24 V/cm of gel width for at least 30 min. Turn off power supply, flush wells, load sample and run gel at same voltage as pre-run.</li>
			<li>Load the sample and run the PAGE until the bromophenol blue has just exited the gel. The desired product will migrate close to the xylene cyanol.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Staining, Visualization, and Excision
	<ol>
		<li>Disassemble gel apparatus and place gel in clean tray with 1x&#160;running buffer (TBE), 1x&#160;sybr-gold, and rock for 15 min.</li>
		<li>Remove gel from staining dish and visualize on UV transilluminator box covered with clean plastic wrap. The ligation product should be around 45 nucleotides in length.</li>
		<li>Excise the region containing the ligation product with a clean razor blade and place in 0.5 ml micro-centrifuge tube.</li>
	</ol>
	</li>
	<li>Product Isolation and Precipitation
	<ol>
		<li>Pierce bottom of 0.5 ml microcentrifuge tube with a 30 G&#160;needle and place the pierced tube in a second clean, low-retention, 1.5 ml microcentrifuge tube. Centrifuge for 5 min at ~15,000 x g.</li>
		<li>Visually confirm that the entire gel slice has moved through the hole in the inner tube. If necessary, use a clean pipette tip to push some gel fragments closer to the hole.</li>
		<li>Discard the empty 0.5 ml tube and add 400 &#956;l of high-salt column buffer (HSCB 400 mM NaCl, 25 mM Tris-HCl pH 7.5, 0.1% SDS) to the acrylamide slurry. Rock tube(s) at 4 &#176;C overnight.</li>
		<li>Transfer slurry to a 0.22 micron cellulose acetate spin column using a wide-bore pipette tip. Centrifuge for 3 min at room temperature at ~15,000 x g to collect all liquid away from acrylamide gel matrix.</li>
		<li>Transfer the liquid to a clean, low-retention, 1.5 ml tube containing 1 &#956;l of 20 mg/ml glycogen. Add an equal volume of isopropanol and 1/10th volume sodium acetate. Mix solution by inverting tube several times and precipitate in -80 &#176;C freezer for 20 min.</li>
		<li>Centrifuge at full speed at 4 &#176;C for 20 min to pellet the nucleic acid. Remove supernatant and wash in 70% ethanol. Air dry pellet for 10 min at room temperature and proceed directly to 5&#8217; ligation step.</li>
	</ol>
	</li>
</ol>
3. Linker Ligation to 5&#8217; End of miRNA-3&#8217; Linker Hybrid
<ol>
	<li>To pelleted nucleic acid, add the following components: 2 &#181;l PEG 8000 (50% w/v), 0.5 &#181;l 10x&#160;RNA Ligase Buffer, 0.5 &#181;l ATP (10 mM), 0.5 &#181;l NN-RNA oligo (100 &#181;M), and H2O to a final volume of 4 &#181;l. 
	NOTE: The composition of the &#8216;NN-RNA oligo&#8217; is as follows 5&#8217;-inverted dideoxy-TCUACAGUCCGACGAUCNN3&#8217; and was purified via HPLC by the manufacturer.</li>
	<li>Mix this sample by pipetting up and down repeatedly. Do this for some time (up to 5 min) to ensure that the pellet has been completely re-solubilized. If re-solubilization is problematic, heat the sample briefly (3-5 min) to 37 &#176;C and resume pipetting to mix.</li>
	<li>Once the pellet is back in solution, transfer the contents to a clean PCR tube containing 1 &#956;l of a 1:1 mixture of RNase inhibitor and T4 RNA Ligase 1 (5 units). Again mix by pipetting up and down.</li>
	<li>Incubate the reaction in a thermal cycler at 37 &#176;C for 4 hr. Proceed immediately to cDNA synthesis.</li>
</ol>
4. Reverse Transcription/cDNA Synthesis
<ol>
	<li>Add the following components to the 5&#8217; Ligation reaction: 2 &#181;l 5x&#160;RT buffer, 0.75 &#181;l 100 mM DTT, 1 &#181;l Illumina RT Primer 5&#8217;-GCCTTGGCACCCGAGAATTCCA-3&#8217; (1 &#181;M), and 0.5 &#181;l dNTPs (10 mM).</li>
	<li>Mix by pipetting and heat to 65 &#176;C for 5 min. Then slowly cool on the bench top.</li>
	<li>Once reaction has cooled to room temperature, place tube on ice and add 0.5 &#956;l each of reverse transcriptase enzyme and RNase inhibitor. Mix by pipetting and place in thermal cycler for the extension step indicated in the manufacturer&#8217;s instructions.</li>
	<li>Once complete add 5 &#956;l of nuclease-free H2O to bring the final volume of the cDNA library up to 15 &#956;l.</li>
</ol>
5. Library Preparation
<ol>
	<li>For the indexing PCR, assemble the following reaction on ice: 6 &#181;l 5x&#160;Phusion Buffer, 1 &#181;l dNTPs (10 mM), 0.9 &#181;l&#160;DMSO, 0.75 &#181;l 5&#8217; Primer (25 &#181;M), 0.75 &#181;l 3&#8217; Primer (25 &#181;M), 3 &#181;l cDNA library (variable concentration depending on the amount of the input), 0.5 &#181;l Phusion Polymerase (1 unit), nuclease-free H2O to a final volume of 30 &#956;l. Choose the primer sequences to assure compatibility with the template and the sequencing platform of choice.
	<ol>
		<li>Run the PCR reaction with the following settings: initial denaturation at 98 &#176;C for 3 min, for the first 4 cycles, denature 98 &#176;C for 15 sec, anneal at 50 &#176;C for 15 sec, extend at 72 &#176;C for 15 sec. 
		NOTE: Depending on the amount of the input material, vary the total PCR cycle numbers. Typically, for an input of 1-2 &#181;g of total RNA, 13 total PCR cycles are sufficient to generate the miR-Seq library.</li>
		<li>Carry out subsequent cycles, e.g., cycles 5-12, in a two-step manner where the annealing and extension cycles are combined and carried out at 70 &#176;C for 30 sec&#160;and the denaturation is again 98 &#176;C for 15 sec. 
		NOTE: The reaction volume is 30 &#956;l to permit pausing the PCR program and remove a 10 &#956;l aliquot at pre-determined cycle numbers to monitor for appearance of desired product. Typically, cycles 10, 13, and 16 are chosen for testing from a total RNA sample.</li>
	</ol>
	</li>
	<li>Prepare an 8% non-denaturing acrylamide gel by mixing 2 ml of 40% acrylamide, 1 ml of 10x&#160;TBE, and H2O to 10 ml. Then add 100 &#956;l of 10% APS and 10 &#956;l of TEMED. Pour gel and let set.
	<ol>
		<li>Pre-run the gel at 12 V/cm of gel width for 30 min. Flush wells. Add 1 &#956;l of 10x&#160;loading buffer (15% Ficoll-400, 66 mM EDTA, 20 mM Tris-HCl pH 8.0, 0.1% (w/v) SDS, 0.01% (w/v) bromophenol blue), add 10 &#956;l of sample to each well of dimensions 5 mm x 1 mm x 15 mm. Typically, each well can load up to 30 &#956;l of samples. Run the sample gel at 10 V/cm of gel width in 1x&#160;TBE buffer until the bromophenol blue dye just runs out of the bottom of the gel.</li>
		<li>Stain and visualize gel as described in step 2.3.</li>
		<li>Excise the 146 base pair band and elute overnight in 0.25 ml of HSCB in a fashion similar to what is described in section 2.4. Precipitate DNA in an equal volume of isopropanol and 1/10th volume sodium acetate. Wash pellet in 70% ethanol and resuspend in TE buffer.</li>
		<li>Quantify the library using picogreen according to manufacturer&#8217;s recommendations.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Hafner, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-ligase-dependent+biases+in+miRNA+representation+in+deep-sequenced+small+RNA+cDNA+libraries.">RNA-ligase-dependent biases in miRNA representation in deep-sequenced small RNA cDNA libraries.</a> RNA. 17 (9), 1697-1712 (2011).</li><li>Zhang, Z., Lee, J. E., Riemondy, K., Anderson, E. M., Yi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+RNA+cloning+enables+accurate+quantification+of+miRNA+expression+by+deep+sequencing.">High-efficiency RNA cloning enables accurate quantification of miRNA expression by deep sequencing.</a> Genome Biology. 14 (10), 109 (2013).</li><li>Consortium, T. E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ENCODE+(ENCyclopedia+Of+DNA+Elements)+Project.">The ENCODE (ENCyclopedia Of DNA Elements) Project.</a> Science. 306 (5696), 636-640 (2004).</li><li>Consortium, T. E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+encyclopedia+of+DNA+elements+in+the+human+genome.">An integrated encyclopedia of DNA elements in the human genome.</a> Nature. 489 (7414), 57-74 (2012).</li><li>Linsen, S. E. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limitations+and+possibilities+of+small+RNA+digital+gene+expression+profiling.">Limitations and possibilities of small RNA digital gene expression profiling.</a> Nature Methods. 6 (7), 474-476 (2009).</li><li>Jayaprakash, A. D., Jabado, O., Brown, B. D., Sachidanandam, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+remediation+of+biases+in+the+activity+of+RNA+ligases+in+small-RNA+deep+sequencing.">Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing.</a> Nucleic Acids Research. 39 (21), 141 (2011).</li><li>McLaughlin, L. W., Romaniuk, E., Romaniuk, P. J., Neilson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effect+of+Acceptor+Oligoribonucleotide+Sequence+on+the+T4+RNA+Ligase+Reaction.">The Effect of Acceptor Oligoribonucleotide Sequence on the T4 RNA Ligase Reaction.</a> European Journal of Biochemistry. 125 (3), 639-643 (1982).</li><li>Zhuang, F., Fuchs, R. T., Sun, Z., Zheng, Y., Robb, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+bias+in+T4+RNA+ligase-mediated+3&#8242;-adapter+ligation.">Structural bias in T4 RNA ligase-mediated 3&#8242;-adapter ligation.</a> Nucleic Acids Research. 40 (7), 54 (2012).</li><li>Harrison, B., Zimmerman, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymer-stimulated+ligation:+enhanced+ligation+of+oligo-+and+polynucleotides+by+T4+RNA+ligase+in+polymer+solutions.">Polymer-stimulated ligation: enhanced ligation of oligo- and polynucleotides by T4 RNA ligase in polymer solutions.</a> Nucleic Acids Research. 12 (21), 8235-8251 (1984).</li><li>Torchia, C., Takagi, Y., Ho, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Archaeal+RNA+ligase+is+a+homodimeric+protein+that+catalyzes+intramolecular+ligation+of+single-stranded+RNA+and+DNA.">Archaeal RNA ligase is a homodimeric protein that catalyzes intramolecular ligation of single-stranded RNA and DNA.</a> Nucleic Acids Research. 36 (19), 6218-6227 (2008).</li><li>Lau, N. C., Lim, L. P., Weinstein, E. G., Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Abundant+Class+of+Tiny+RNAs+with+Probable+Regulatory+Roles+in+Caenorhabditis+elegans.">An Abundant Class of Tiny RNAs with Probable Regulatory Roles in Caenorhabditis elegans.</a> Science. 294 (5543), 858-862 (2001).</li><li>Yi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DGCR8-dependent+microRNA+biogenesis+is+essential+for+skin+development.">DGCR8-dependent microRNA biogenesis is essential for skin development.</a> Proceedings of the National Academy of Sciences. 106 (2), 498-502 (2009).</li><li>Leung, A. K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+identification+of+Ago2+binding+sites+from+mouse+embryonic+stem+cells+with+and+without+mature+microRNAs.">Genome-wide identification of Ago2 binding sites from mouse embryonic stem cells with and without mature microRNAs.</a> Nature Structural &amp; Molecular Biology. 18 (2), 237-244 (2011).</li></ol>

The authors declare no competing financial interests.

The methodology described herein makes use of several key variables to maximize ligation efficiencies, namely high concentrations of PEG, use of randomized linkers, and high concentration of linkers2,6,9. This approach permits reliably quantitative sequencing libraries from total RNA samples2. We have conducted multiple titrations of input RNA and have concluded that the preceding methodology is best suited for total RNA amounts in the 1-8 &#956;g range (data not shown). When amounts in the 10-500 n...

High-throughput sequencing based methodologies have been widely applied to many biological samples in recent years greatly expanding our understanding of the molecular complexity of biological systems3,4. However, preparation of RNA samples for high-throughput sequencing often imparts specific biases inherent to the employed methodology, limiting the potential utility of these powerful techniques. These method specific biases have been well documented for ligation-based, small-RNA library preparations1,2,5,6. These biases result in 1,000-fold variation in reads numbers for equimolar synthetic miRNAs, making inference of miRNA abundance from sequencing data wildly variable and error prone.
Studies focusing on the properties of phage-derived T4 RNA ligases have documented that the enzymes exhibit nucleotide-based preferences7, which manifest as biased libraries in high-throughput sequencing experiments1,2,8. In order to minimize the biases imparted by RNA ligases, multiple strategies have been employed; macromolecular crowding9, randomizing the nucleotide sequence on the adapter which is proximal to the ligation site6, and employing high concentrations of ligation adapter2. Through a combination of these three approaches we have developed a work-flow for unbiased preparation of small RNA libraries compatible for high-throughput sequencing (Figure 1). For direct comparisons between current protocols and our optimized method, please refer to our recent report2. This optimized method yields ligation efficiencies of greater than 95% at both 3&#8217; and 5&#8217; steps and permits the unbiased ligation of small RNA molecules from synthetic and biological samples2.

<table><tbody><tr><td>3&#039; Linker (5&#039; phosphorylated, 3&#039; blocked)</td><td>Integrated DNA Technologies</td><td>custom</td><td></td></tr><tr><td>5&#039; Linker</td><td>Integrated DNA Technologies</td><td>custom</td><td>5&#039; blocked, HPLC purified</td></tr><tr><td>T4RNL2 (1-249 K227Q)</td><td>New England Biolabs</td><td>M0351S</td><td>Specialized for ligation of pre-adenylated DNA adapters</td></tr><tr><td>10x Ligation buffer (without ATP)</td><td>New England Biolabs</td><td>Included with M0351S</td><td></td></tr><tr><td>10x Ligation buffer (with ATP)</td><td>New England Biolabs</td><td>Included with M0204L</td><td></td></tr><tr><td>RNaseOUT</td><td>Invitrogen</td><td>10777-019</td><td></td></tr><tr><td>Polyethylene glycol (mol. Wt. 8,000)</td><td>New England Biolabs</td><td>Included with M0204L</td><td></td></tr><tr><td>Nuclease-free water</td><td>Ambion</td><td>AM9937</td><td>We have found water collected from a distillation apparatus to be of equvalent quality.</td></tr><tr><td>T4RNL1</td><td>New England Biolabs</td><td>M0204L</td><td></td></tr><tr><td>Superscript III RT kit</td><td>Invitrogen</td><td>18080-051&amp;nbsp;</td><td></td></tr><tr><td>Phusion PCR kit</td><td>New England Biolabs</td><td>M0530S</td><td></td></tr><tr><td>Illumina RP1 primer</td><td>Integrated DNA Technologies</td><td>custom</td><td>Sequence information available from Illumina</td></tr><tr><td>Illumina RT primer</td><td>Integrated DNA Technologies</td><td>custom</td><td>Sequence information available from Illumina</td></tr><tr><td>Illumina index primer(s)</td><td>Integrated DNA Technologies</td><td>custom</td><td>Sequence information available from Illumina</td></tr><tr><td>40% Acrylamide</td><td>Fisher Scientific</td><td>BP14081</td><td></td></tr><tr><td>Urea</td><td>Sigma Aldrich</td><td>U6504</td><td></td></tr><tr><td>Ammonium persulfate</td><td>Sigma Aldrich</td><td>A3678</td><td></td></tr><tr><td>Tetramethyethylenediamine (TEMED)</td><td>Sigma Aldrich</td><td>T9281</td><td></td></tr><tr><td>2x Denaturing RNA loading buffer</td><td>New England Biolabs</td><td>Included with M0351S</td><td></td></tr><tr><td>Razor blades</td><td>VWR</td><td>55411-050</td><td></td></tr><tr><td>SpinX Centricon tubes</td><td>Costar</td><td>CLS8161</td><td></td></tr><tr><td>Low retention microfuge tubes</td><td>Fisher Scientific</td><td>02-681-320</td><td></td></tr><tr><td>Sybr Gold</td><td>Invitrogen</td><td>S-11494</td><td></td></tr><tr><td>Adenylation kit</td><td>New England Biolabs</td><td>E2610L</td><td></td></tr></tbody></table>

highly efficient ligation of small rna molecules for microrna quantitation by high-throughput sequencing

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide resolution. This technique captures miRNAs by attaching 3&#8217; and 5&#8217; oligonucleotide adapters to miRNA molecules and allows de novo miRNA discovery. Coupling with powerful next-generation sequencing platforms, miR-Seq has been instrumental in the study of miRNA biology. However, significant biases introduced by oligonucleotide ligation steps have prevented miR-Seq from being employed as an accurate quantitation tool. Previous studies demonstrate that biases in current miR-Seq methods often lead to inaccurate miRNA quantification with errors up to 1,000-fold for some miRNAs1,2. To resolve these biases imparted by RNA ligation, we have developed a small RNA ligation method that results in ligation efficiencies of over 95% for both 3&#8217; and 5&#8242; ligation steps. Benchmarking this improved library construction method using equimolar or differentially mixed synthetic miRNAs, consistently yields reads numbers with less than two-fold deviation from the expected value. Furthermore, this high-efficiency miR-Seq method permits accurate genome-wide miRNA profiling from in vivo total RNA samples2.

The anticipated results for the preceding method should initially be observation of a single nucleotide shift (increase) in the size of the DNA oligonucleotide that was subject to adenylation by Mth RNA ligase (Figure 2). Following 3&#8217; ligation, visualization of the acrylamide gel indicates (see Figure 3) sharp high molecular weight bands evident in the 100-300 nucleotide region of the gel. This indicates that the total RNA sample being used is of high quality (not degraded...

Watch this Scientific Journal Video about Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing at JoVE.com

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

highly-efficient-ligation-small-rna-molecules-for-microrna

Research

JoVE Journal

Biology

11.8K Views.  University of Colorado, Boulder. MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs. 

Video: Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the ...

Bioengineering

A Complete Pipeline for Isolating and Sequencing MicroRNAs, and Analyzing Them Using Open Source Tools

Half of all human transcripts are thought to be regulated by microRNAs. Therefore, quantifying microRNA expression can reveal underlying mechanisms in disease states and provide therapeutic targets and biomarkers. Here, we detail how to accurately quantify microRNAs. Briefly, this method describes isolating microRNAs, ligating them to adaptors suitable for high-throughput sequencing, amplifying the final products, and preparing a sample library. Then, we explain how to align the obtained sequencing reads to microRNA hairpins, and quantify, normalize, and calculate their differential expression. Versatile and robust, this combined experimental workflow and bioinformatic analysis enables users to begin with tissue extraction and finish with microRNA quantification.

Here, we describe a step-by-step strategy for isolating small RNAs, enriching for microRNAs, and preparing samples for high-throughput sequencing. We then describe how to process sequence reads and align them to microRNAs, using open source tools.

Half of all human transcripts are thought to be regulated by microRNAs. Therefore, quantifying microRNA expression can reveal underlying mechanisms in ...

Genetics

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as plant microRNAs (miRNAs) carry a 2&#39;-O-methyl (2&#39;-OMe) modification at their 3&#39;&#160;terminal nucleotide. This modification adds another difficulty as it inhibits 3&#39;&#160;adapter ligation. We previously demonstrated that modified versions of the &#39;TruSeq (TS)&#39;&#160;protocol have less bias and an improved detection of 2&#39;-OMe RNAs. Here we describe in detail protocol &#39;TS5&#39;, which showed the best overall performance. TS5 can be followed either using homemade reagents or reagents from the TS kit, with equal performance.

We present a detailed small RNA library reparation protocol with less bias than standard methods and an increased sensitivity for 2&#39;-O-methyl RNAs. This protocol can be followed using homemade reagents to save cost or using kits for convenience.

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as ...

Identification of RNAs Engaged in Direct RNA-RNA Interaction with a Long Non-Coding RNA

The growing role attributed nowadays to long non-coding RNAs (lncRNA) in physiology and pathophysiology makes it crucial to characterize their interactome by identifying their molecular partners, DNA, proteins and/or RNAs. The latter can interact with lncRNA through networks involving proteins, but they can also be engaged in direct RNA/RNA interactions. We, therefore, developed an easy-to-use RNA pull-down procedure that allowed identification of RNAs engaged in direct RNA/RNA interaction with a lncRNA using psoralen, a molecule that cross-links only RNA/RNA interactions. Bioinformatics modeling of the lncRNA secondary structure allowed the selection of several specific antisense DNA oligonucleotide probes with a strong affinity for regions displaying a low probability of internal base pairing. Since the specific probes that were designed targeted accessible regions throughout the length of the lncRNA, the RNA-interaction zones could be delineated in the sequence of the lncRNA. When coupled with a high throughput RNA sequencing, this protocol can be used for the whole direct RNA interactome studies of a lncRNA of interest.

An easy-to-use RNA pull-down protocol is designed for the identification of RNAs engaged in direct RNA/RNA interaction with a long non-coding RNA. The protocol uses psoralen as a fixative to cross-link only RNA/RNA interactions and provides the whole direct RNA interactome of a long non-coding RNA when coupled with RNA sequencing.

The growing role attributed nowadays to long non-coding RNAs (lncRNA) in physiology and pathophysiology makes it crucial to characterize their interactome ...

RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and animals. Most miRNAs act as negative switches of gene expression targeting key genes. In plants, primary miRNAs (pri-miRNAs) transcripts are generated by RNA polymerase II, and they form varying lengths of stable stem-loop structures called pre-miRNAs. An endonuclease, Dicer-like1, processes the pre-miRNAs into miRNA-miRNA* duplexes. One of the strands from miRNA-miRNA* duplex is selected and loaded onto Argonaute 1 protein or its homologs to mediate the cleavage of target mRNAs. Although miRNAs are key signaling molecules, their detection is often carried out by less than optimal PCR-based methods instead of a sensitive northern blot analysis. We describe a simple, reliable, and extremely sensitive northern method that is ideal for the quantification of miRNA levels with very high sensitivity, literally from any plant tissue. Additionally, this method can be used to confirm the size, stability and the abundance of miRNAs and their precursors.

This method demonstrates use of the northern hybridization technique to detect miRNAs from total RNA extract.

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding, ~21 nt small RNAs involved in the regulation of gene expression in both plants and ...

Biochemistry

Analyzing tRNA Dynamics Using AQRNA-seq

AQRNA-seq for Quantifying Small RNAs

AQRNA-seq provides a direct linear relationship between sequencing read counts and small RNA copy numbers in a biological sample, thus enabling accurate quantification of the pool of small RNAs. The AQRNA-seq library preparation procedure described here involves the use of custom-designed sequencing linkers and a step for reducing methylation RNA modifications that block reverse transcription processivity, which results in an increased yield of full-length cDNAs. In addition, a detailed implementation of the accompanying bioinformatics pipeline is presented. This demonstration of AQRNA-seq was conducted through a quantitative analysis of the 45 tRNAs in Mycobacterium bovis BCG harvested on 5 selected days across a 20-day time course of nutrient deprivation and 6 days of resuscitation. Ongoing efforts to improve the efficiency and rigor of AQRNA-seq will also be discussed here. This includes exploring methods to obviate gel purification for mitigating primer dimer issues after PCR amplification and to increase the proportion of full-length reads to enable more accurate read mapping. Future enhancements to AQRNA-seq will be focused on facilitating automation and high-throughput implementation of this technology for quantifying all small RNA species in cell and tissue samples from diverse organisms.

Author Spotlight: AQRNA-seq Role in Mapping Small RNAs and Unraveling Protein Translation Mechanisms

Absolute quantification RNA sequencing (AQRNA-seq) is a technology developed to quantify the landscape of all small RNAs in biological mixtures. Here, both the library preparation and data processing steps of AQRNA-seq are demonstrated, quantifying changes in the transfer RNA (tRNA) pool in Mycobacterium bovis BCG during starvation-induced dormancy.

AQRNA-seq provides a direct linear relationship between sequencing read counts and small RNA copy numbers in a biological sample, thus enabling accurate ...

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...

Developmental Biology

Genome-wide Analysis of HDAC Inhibitor-mediated Modulation of microRNAs and mRNAs in B Cells Induced to Undergo Class-switch DNA Recombination and Plasma Cell Differentiation

Antibody responses are accomplished through several critical B cell-intrinsic processes, including somatic hypermutation (SHM), class-switch DNA recombination (CSR), and plasma cell differentiation. In recent years, epigenetic modifications or factors, such as histone deacetylation and microRNAs (miRNAs), have been shown to interact with B-cell genetic programs to shape antibody responses, while the dysfunction of epigenetic factors has been found to lead to autoantibody responses. Analyzing genome-wide miRNA and mRNA expression in B cells in response to epigenetic modulators is important for understanding the epigenetic regulation of B-cell function and antibody response. Here, we demonstrate a protocol for inducing B cells to undergo CSR and plasma cell differentiation, treating these B cells with histone deacetylase (HDAC) inhibitors (HDIs), and analyzing mRNA and microRNA expression. In this protocol, we directly analyze complementary DNA (cDNA) sequences using next-generation mRNA sequencing (mRNA-seq) and miRNA-seq technologies, mapping of the sequencing reads to the genome, and quantitative reverse transcription (qRT)-PCR. With these approaches, we have defined that, in B cells induced to undergo CSR and plasma cell differentiation, HDI, an epigenetic regulator, selectively modulates miRNA and mRNA expression and alters CSR and plasma cell differentiation.

Epigenetic factors can interact with genetic programs to modulate gene expression and regulate B cell function. By combining in vitro B-cell stimulation, qRT-PCR, and high-throughput microRNA-sequence and mRNA-sequence approaches, we can analyze the epigenetic modulation of miRNA and gene expression in B cells.

Antibody responses are accomplished through several critical B cell-intrinsic processes, including somatic hypermutation (SHM), class-switch DNA ...

Immunology and Infection

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...