Name: retrospective microrna sequencing: complementary dna library preparation protocol using formalin-fixed paraffin-embedded rna specimens
Uploaded: 2018-05-05T13:00:00
Description: Watch this Scientific Journal Video about Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens at JoVE.com

We thank Dr. Thomas Tuschl, head of the laboratory for RNA molecular biology, as well as members of his laboratory for their support and for sharing the technology developed in his laboratory and providing access to the RNAworld pipeline. We also thank Dr. Markus Hafner for sharing his protocol and providing detailed descriptions on all biochemical and enzymatic steps used in his initial procedure.

1. Preparation of All Reagents and Primers
<ol>
	<li>Order all primers and adapters as described in Figure 1.</li>
	<li>Prepare the calibrator cocktail stock, which will be spiked in each individual ligation.
	<ol>
		<li>Resuspend the carrier oligonucleotide (Figure 1) to 0.5 &#181;M with RNase-free water. Resuspend the 10 calibrator oligonucleotides (Figure 1) to 100 &#181;M using the 0.5 &#181;M carrier oligonuc...

<ol>
	<li>Peir&#243;-Chova, L., Pe&#241;a-Chilet, M., L&#243;pez-, J. A., Garc&#237;a-Gimenez, J. L., Alonso-Yuste, E., Burgues, O., 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=High+stability+of+microRNAs+in+tissue+samples+of+compromised+quality.">High stability of microRNAs in tissue samples of compromised quality.</a> Virchows. Arch. 463, 765-774 (2013).</li><li>Klopfleisch, R., Weiss, A. T., Gruber, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excavation+of+a+buried+treasure--DNA,+mRNA,+miRNA+and+protein+analysis+in+formalin+fixed,+paraffin+embedded+tissues.">Excavation of a buried treasure--DNA, mRNA, miRNA and protein analysis in formalin fixed, paraffin embedded tissues.</a> Histol. Histopathol. 26, 797-810 (2011).</li><li>Kakimoto, Y., Kamiguchi, H., Ochiai, E., Satoh, F., Osawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+Stability+in+Postmortem+FFPE+Tissues:+Quantitative+Analysis+Using+Autoptic+Samples+from+Acute+Myocardial+Infarction+Patients.">MicroRNA Stability in Postmortem FFPE Tissues: Quantitative Analysis Using Autoptic Samples from Acute Myocardial Infarction Patients.</a> PLoS One. 10 (6), e0129338 (2015).</li><li>Giricz, O., Reynolds, P. 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Pathol. 62, 84-88 (2009).</li><li>Jang, J. S., Simon, V. A., Feddersen, R. M., Rakhshan, F., Schultz, D. A., Zschunke, M. A., Lingle, W. L., Kolbert, C. P., Jen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+miRNA+expression+analysis+using+fluidigm+microfluidics+dynamic+arrays.">Quantitative miRNA expression analysis using fluidigm microfluidics dynamic arrays.</a> BMC Genomics. 12, 144 (2011).</li><li>Andreasen, D., Fog, J. U., Biggs, W., Salomon, J., Dahslveen, I. K., Baker, A., Mouritzen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+microRNA+quantification+in+total+RNA+from+clinical+samples.">Improved microRNA quantification in total RNA from clinical samples.</a> Methods. 50 (4), S6-S9 (2010).</li><li>Kelly, A. D., Hill, K. E., Correll, M., Wang, Y. 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A., Alvarez-Saavedra, E., Lamb, J., Peck, D., 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=MicroRNA+expression+profiles+classify+human+cancers.">MicroRNA expression profiles classify human cancers.</a> Nature. 435, 834-838 (2005).</li><li>Cho, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs:+potential+biomarkers+for+cancer+diagnosis,+prognosis+and+targets+for+therapy.">MicroRNAs: potential biomarkers for cancer diagnosis, prognosis and targets for therapy.</a> Int. J. Biochem. Cell. Biol. 42, 1273-1281 (2010).</li><li>Hui, A., How, C., Ito, E., Liu, F. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-RNAs+as+diagnostic+or+prognostic+markers+in+human+epithelial+malignancies.">Micro-RNAs as diagnostic or prognostic markers in human epithelial malignancies.</a> BMC Cancer. 11, 500 (2011).</li><li>Tam, S., de Borja, R., Tsao, M. S., McPherson, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+global+microRNA+expression+profiling+using+next-generation+sequencing+technologies.">Robust global microRNA expression profiling using next-generation sequencing technologies.</a> Lab. Invest. 94, 350-358 (2014).</li><li>Loudig, O., Wan, T., Ye, K., Lin, J., Wang, Y., Ramnauth, A., 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=Evaluation+and+Adaptation+of+a+Laboratory+Based+cDNA+Library+Preparation+Protocol+for+Retrospective+Sequencing+of+Archived+MicroRNAs+from+up+to+35-Year-Old+Clinical+FFPE+Specimens.">Evaluation and Adaptation of a Laboratory Based cDNA Library Preparation Protocol for Retrospective Sequencing of Archived MicroRNAs from up to 35-Year-Old Clinical FFPE Specimens.</a> Int J Mol Sci. 18, e627 (2017).</li><li>Kotorashvili, A., Ramnauth, A., Liu, C., Lin, J., Ye, K., Kim, 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=Effective+DNA/RNA+co-extraction+for+analysis+of+microRNAs,+mRNAs,+and+genomic+DNA+from+formalin-fixed+paraffin-embedded+specimens.">Effective DNA/RNA co-extraction for analysis of microRNAs, mRNAs, and genomic DNA from formalin-fixed paraffin-embedded specimens.</a> PLoS One. 7 (4), e34683 (2012).</li><li>Hafner, M., Renwick, N., Farazi, T. A., Mihailovi&#263;, A., Pena, J. T., Tuschl, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barcoded+cDNA+library+preparation+for+small+RNA+profiling+by+next-generation+sequencing.">Barcoded cDNA library preparation for small RNA profiling by next-generation sequencing.</a> Methods. 58 (2), 164-170 (2012).</li><li>Loudig, O., Brandwein-Gensler, M., Kim, R. 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A highly reproducible and robust cDNA library preparation protocol for NGS of small RNAs archived in FFPE RNA specimens is presented in this protocol, which is a modified and optimized version of the procedure described by Hafner et al.22
All steps of this protocol have been optimized for use with older archived and compromised total RNA recovered from FFPE specimens. The key step of this protocol, for processing small amounts of FFPE RNA, resides in the poolin...

miRNAs are remarkably well conserved in formalin-fixed paraffin-embedded (FFPE) specimens1,2,3. Previous work has demonstrated that the expression of these short regulatory non-coding single stranded RNA molecules can be successfully evaluated using total RNA from FFPE samples and provide relevant gene expression data when compared to the original fresh tissues4,5,6,7,8. When compared to large-size messenger RNAs, which have been shown to be critically affected by FFPE tissue processing (formaldehyde, heat, desiccation, etc.), endogenous RNases, and the age of the specimens, the small size of miRNAs (~18&#8211;24 nucleotides) appears to make them resistant to degradation and resilient to long-term storage, also demonstrated through miRNA expression studies that outperform high-throughput mRNA studies in archived specimens9. miRNA expression studies using archived clinical specimens, which have mostly been performed in small-scale analyses, have demonstrated that single or multiplexed quantitative PCR assays, different types of microarray technologies, and most recently NGS can be used to assess the expression of preserved miRNAs after optimization of these assays10,11,12,13,14.
Given that dysregulation of miRNA expression has been associated with the development of a variety of human malignancies and that there is potentially an enormous supply of clinically annotated archived specimens, it has become apparent that these small RNA molecules represent a promising source of potential cancer biomarkers15,16,17,18. The use of a high-throughput gene expression technology such as NGS has the advantage of providing a global evaluation of all miRNA transcripts when compared to targeted technologies such as PCR and/or microarrays19. For this reason, an optimized, affordable, and easily applicable protocol for cDNA library preparation of small RNAs from older archived specimens for NGS was optimized to enable large-scale retrospective studies20.
We previously established a simultaneous RNA/DNA extraction protocol for separate recovery of RNA and DNA from older archived specimens, which we found to outperform contemporary commercial kits21. Using this extraction protocol, to obtain total RNA from FFPE tissues archived for extended period of times, we optimized the preparation of cDNA libraries for NGS of miRNAs preserved in clinical specimens for up to 35 years. Furthermore, in a recently published study where we prepared cDNA libraries from clinically classified ductal carcinoma in situ (DCIS) specimens, we identified differentially expressed miRNAs that were validated by quantitative PCR, which indicated that specific miRNA expression changes may be detectable in DCIS lesions from patients who develop breast cancer when compared to DCIS lesions from patients who do not develop breast cancer.
Considering the cost of commercial kits for preparation of small RNA cDNA libraries, the potential for their discontinuation, as well as the use of copyright/patent-protected reagents that cannot be optimized, we decided to adapt a previously published laboratory-based and kit-free 3&#39; barcoded cDNA library preparation protocol for NGS of small RNAs archived in FFPE specimens, allowing simultaneous analysis of 18 samples22. This protocol provides an ideal and robust step-by-step procedure with visual and technical evaluation checkpoints, which were critical for adaptation to FFPE RNA specimens, and has a strong potential for application to other sources of compromised or difficult to use RNA material. The original protocol&#39;s applicability was improved by replacing radioactively labeled size markers with fluorescent (e.g., SYBR Gold) detectable RNA size markers used during selection of ligated libraries on large polyacrylamide gels. This optimized protocol relies on the ligation of 3&#39; barcoded adapters to 18 individual FFPE RNA specimens, which are then pooled together to undergo 5&#39; adapter ligation, reverse-transcription, and a pilot PCR analysis for tailored amplification of the final cDNA library prior to large-scale PCR amplification, purification, and NGS on a high throughput sequencer.

<table>
	<tbody>
		<tr>
			<td>1% Triton x-100</td>
			<td>Invitrogen</td>
			<td>HFH10</td>
			<td></td>
		</tr>
		<tr>
			<td>10mM ATP</td>
			<td>Ambion</td>
			<td>AM8110G</td>
			<td></td>
		</tr>
		<tr>
			<td>10X dNTPs</td>
			<td>Ambion</td>
			<td>AM8110G</td>
			<td></td>
		</tr>
		<tr>
			<td>10x TBE</td>
			<td>Thermofisher Scientific</td>
			<td>15581044</td>
			<td></td>
		</tr>
		<tr>
			<td>14M Mercaptoethanol</td>
			<td>Sigma</td>
			<td>O3445I-100</td>
			<td></td>
		</tr>
		<tr>
			<td>20 nt ladder</td>
			<td>Jena Bioscience</td>
			<td>M-232S</td>
			<td></td>
		</tr>
		<tr>
			<td>20mg/ml Bovine Serum Albumine</td>
			<td>Sigma</td>
			<td>B8894-5ML</td>
			<td></td>
		</tr>
		<tr>
			<td>50X Titanium Taq</td>
			<td>Clontech Laboratories</td>
			<td>639208</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Persulfate</td>
			<td>Fisher Scientific</td>
			<td>7727-54-0</td>
			<td></td>
		</tr>
		<tr>
			<td>BRL Vertical Gel Electrophoresis System with glass plates and combs</td>
			<td>GIBCO</td>
			<td>V16</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma</td>
			<td>D9170-5VL</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf microcentrifuge 5424R</td>
			<td>USA scientific</td>
			<td>4054-4537Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppndorf Thermomixer</td>
			<td>USA scientific</td>
			<td>4053-8223Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand&#8482; Siliconized Low-Retention Microcentrifuge Tubes 1.5ml</td>
			<td>Fisher Scientific</td>
			<td>02-681-320</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Breaker Tube 0.5 ml</td>
			<td>IST Engineering Inc,</td>
			<td>3388-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel electrophoresis apparatus 7cm x10cm- Mini-sub Cell GT with gel trays and combs</td>
			<td>Biorad</td>
			<td>1704446</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycoblue</td>
			<td>Ambion</td>
			<td>AM9516</td>
			<td></td>
		</tr>
		<tr>
			<td>Jersey-Cote</td>
			<td>LabScientific, Inc</td>
			<td>1188</td>
			<td></td>
		</tr>
		<tr>
			<td>KcL 2M</td>
			<td>Ambion</td>
			<td>AM9640G</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2 1M</td>
			<td>Ambion</td>
			<td>AM9530G</td>
			<td></td>
		</tr>
		<tr>
			<td>Minifuge dual rotor personal centrifuge</td>
			<td>USA scientific</td>
			<td>2641-0016</td>
			<td></td>
		</tr>
		<tr>
			<td>Model V16 polyacrylamide gel electrophoresis apparatus, glasses, combs, and spacers</td>
			<td>Ciore Life Science</td>
			<td>21070010</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligonucleotides</td>
			<td>IDT</td>
			<td>Defined during order</td>
			<td></td>
		</tr>
		<tr>
			<td>Owl EasyCast B2 mini electrophoresis system- with gel trays and combs</td>
			<td>Thermofisher Scientific</td>
			<td>B2</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiaquick Gel Extraction kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td></td>
		</tr>
		<tr>
			<td>Restriction enzyme PmeI</td>
			<td>NEB</td>
			<td>R0560S</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase-free water</td>
			<td>Ambion</td>
			<td>AM9932</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe Imager 2.0</td>
			<td>Life Technologies</td>
			<td>G6600</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe Imager 2.0 blue light transilluminator</td>
			<td>Thermofisher</td>
			<td>G6600</td>
			<td></td>
		</tr>
		<tr>
			<td>SeaKem LE agarose</td>
			<td>Lonza</td>
			<td>50002</td>
			<td></td>
		</tr>
		<tr>
			<td>Superscript III reverse transcription kit</td>
			<td>Invitrogen</td>
			<td>18080-044</td>
			<td></td>
		</tr>
		<tr>
			<td>SybrGold</td>
			<td>Life Technologies</td>
			<td>S11494</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 RNA Ligase 1</td>
			<td>NEB</td>
			<td>M0204S</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 RNA Ligase 2 Truncated K227Q</td>
			<td>NEB</td>
			<td>0351L</td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Fisher Scientific</td>
			<td>O3446I-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Themocycler with heated lid</td>
			<td>Applied Biosystem</td>
			<td>4359659</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris 1M pH 7.5</td>
			<td>Invitrogen</td>
			<td>15567027</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris 1M pH8.0</td>
			<td>Ambion</td>
			<td>AM9855G</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Sequagel system concentrate, diluent, and buffer</td>
			<td>National Diagnostics</td>
			<td>EC-833</td>
			<td></td>
		</tr>
	</tbody>
</table>

retrospective microrna sequencing: complementary dna library preparation protocol using formalin-fixed paraffin-embedded rna specimens

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of cancer development. By using non-invasive or pre-malignant lesions from patients who later develop invasive disease, gene expression analyses may help identify early molecular alterations that predispose to cancer risk. It has been well described that nucleic acids recovered from FFPE tissues have undergone severe physical damage and chemical modifications, which make their analysis difficult and generally requires adapted assays. MicroRNAs (miRNAs), however, which represent a small class of RNA molecules spanning only up to ~18&#8211;24 nucleotides, have been shown to withstand long-term storage and have been successfully analyzed in FFPE samples. Here we present a 3&#39; barcoded complementary DNA (cDNA) library preparation protocol specifically optimized for the analysis of small RNAs extracted from archived tissues, which was recently demonstrated to be robust and highly reproducible when using archived clinical specimens stored for up to 35 years. This library preparation is well adapted to the multiplex analysis of compromised/degraded material where RNA samples (up to 18) are ligated with individual 3&#39; barcoded adapters and then pooled together for subsequent enzymatic and biochemical preparations prior to analysis. All purifications are performed by polyacrylamide gel electrophoresis (PAGE), which allows size-specific selections and enrichments of barcoded small RNA species. This cDNA library preparation is well adapted to minute RNA inputs, as a pilot polymerase chain reaction (PCR) allows determination of a specific amplification cycle to produce optimal amounts of material for next-generation sequencing (NGS). This approach was optimized for the use of degraded FFPE RNA from specimens archived for up to 35 years and provides highly reproducible NGS data.

As described in the method here, a total of 18 individual FFPE RNA samples (100 ng each) are set up in separate tubes to undergo 3&#39; adenylated barcoded oligonucleotide T4 ligation overnight. The next day, the enzymatic reactions are heat-deactivated, combined, and precipitated in a single tube. The RNA pellet is resuspended and the ligated RNA molecules are separated on a 15% denaturing polyacrylamide gel (PAGE), where RNA oligonucleotide size markers that migrated in adjacent wells o...

Watch this Scientific Journal Video about Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens at JoVE.com

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

Formalin-fixed paraffin-embedded specimens represent a valuable source of molecular biomarkers of human diseases. Here we present a laboratory-based cDNA library preparation protocol, initially designed with fresh frozen RNA, and optimized for the analysis of archived microRNAs from tissues stored up to 35 years.

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of ...

The overall goal of this CDNA Library preparation is to enable successful sequencing of small RNAs that have been stored in formal and fixed paraffin-embedded tissues. This method is very useful for analyzing formal and fixed paraffin-embedded specimens. You extract RNA from up to 18 specimens, you analyze the content of all of these and obtain a profile of all of the microRNase for all the specimens.To begin the protocol, add 8.5 microliters of previously-prepared ligation master mix to each of the 18 individually aliquoted FFPERNA samples. Gently flick the tubes, and centrifuge for two seconds. After centrifuging, store the tubes on ice.Use a three microliter pipette to transfer one microliter of each adapter, to the corresponding FFPERNA samples containing RNA and ligation master mix. Do not pipette up and down, and change tips between FFPERNA samples. Close each tube, flick them to mix, and centrifuge them for two seconds.Set the tubes on ice. Denature the reactions by placing the tubes on a heat block at 90 degrees celsius for one minute, and then immediately place them on ice. Next, use a three microliter pipette to transfer one microliter of the diluted ligation enzyme into each of the 18 RNA samples.Set the ligations on ice, and place them in a four degrees celsius cold room for an overnight incubation of 18 hours. Place the tubes on a heat block for one minute at 90 degrees celsius to deactivate the ligation reaction and then return them to ice for at least two minutes to cool down. Transfer 1.2 microliters of the precipitation master mix into each tube.Add 63 microliters of 100%ethanol. Close each tube, flick the tubes to mix, and centrifuge them for two seconds. Place the tubes on ice.Combine the contents of all 18 tubes into a single 1.5 milliliter siliconized tube and invert three times to mix. Centrifuge the tubes for two seconds, and place them on ice for 60 minutes to precipitate. After precipitation, centrifuge the 1.5 milliliter tube with the pooled ligations.Next, remove the supernatant with a one milliliter pipette tip but leave some liquid at the bottom. Tilt the tube, and use a 20 microliter pipette to remove the remaining supernatant. Vacuum-suction the supernatant with a Pasteur pipette with a 10 microliter tip, then resuspend the RNA pellet in 20 microliters of RNase-free water by flicking the tube.Add 20 microliters of PAA gel loading solution to resuspend the RNA. Flick the tube, centrifuge the tube for two seconds at room temperature, and set the tube on ice. Replace the previously prepared 0.5 XTBE of the gel apparatus with fresh 0.5 XTBE, and load the latter, size markers, and ligated RNase in the center of the gel, leaving two empty wells on both sides.Then run the gel. After running the gel, uncast the 15%page by removing one of the glass plates. Lightly spray the gel sitting on the glass plate with 10 microliters of fluorescent dye solution and 25 milliliters of 0.5 XTBE, and let it sit flat for five minutes in the dark.Lay the glass with the gel on a blue-light transilluminator, and align both 19 nucleotide and 24 nucleotide size markers with a ruler to direct the excision of the ligated small RNAs. Then, excise the gel. Place the excised gel in a 0.5 milliliter tube designed to fragment gel slices, securely positioned into a 1.5 milliliter siliconized microcentrifuge tube.Centrifuge the gel at 16 thousand times G for three minutes at room temperature. Resuspend the fragmented gel with 300 microliters of 400 millimolar sodium chloride solution. Close the tube, and seal it with paraffin film.Agitate the contents on a thermo mixer at 11 hundred RPM in a four degrees celsius cold room for an overnight incubation. The next day, retrieve the tube from the thermo mixer and use a one milliliter pipette to transfer the solution onto a five micrometer filter tube, inserted into a 1.5 milliliter siliconized RNase-free collection tube. Centrifuge the tube for three minutes at 23 hundred times G at room temperature.Discard the filter tube, add 950 microliters of 100%ethanol to the collection tube containing the filtered solution, and invert the tube to mix. Centrifuge the tube for two seconds, and place it on ice for 60 minutes. Then, precipitate the RNA pellet.Next, dry the RNA pellet by opening the cap and carefully removing the supernatant with a one milliliter pipette, while leaving some liquid at the bottom. Use a 20 microliter pipette to remove all of the remaining supernatant without touching the pellet. Tilt the tube so any remaining solution moves to the opposite side of the pellet.Use a Pasteur pipette to aspirate the supernatant and dry the pellet using a vacuum. Resuspend the RNA pellet in 5.6 microliters of RNase-free water. Defrost the reverse transcription reagents on ice for 15 minutes.Set up a reverse transcription reaction by adding three microliters of 5X buffer, 4.2 microliters of 10X dioxynucleotide triphosphate, or DNTPs, and 1.5 microliter of dithiothreitol, or DTT. Gently flick the tube to mix the reaction, and centrifuge for two seconds to collect the solution. After the reaction is on a heat block at 90 degrees celsius for exactly 30 seconds, transfer it directly to a thermo mixer at 50 degrees celsius for two minutes to equalize the temperature.Add 0.75 microliters of the reverse transcription enzyme directly into the solution. Flick gently to mix, and set the tube back on the thermo mixer at 50 degrees celsius for 30 minutes immediately. After 30 minutes, transfer the tube to a heat block at 95 degrees celsius for one minute to stop the reverse transcription.Add 95 microliters of RNase-free water directly to the reverse transcription. Flick the tube to mix, and set it directly on ice for two minutes. Assemble the pilot PCR reaction and set up two PCR cycles on the thermo cycler.Set up the pilot PCR reaction on the thermo cycler and start file one. At the end of file one, use a 20 microliter pipette to transfer 12 microliters from the pilot PCR reaction into 1.5 milliliter microcentrifuge tube containing three microliters of 5X gel loading dye. Mix by pipetting up and down.And label it 10 cycles. Close the pilot PCR tube and start file two on the thermo cycler. At the end of file two, use a 20 microliter pipette to transfer 12 microliters of solution into a 1.5 milliliter microcentrifuge tube with three microliters of 5X gel loading dye, and label it 12 cycles.Repeat the steps above to collect 12 microliters of PCR products from the pilot PCR reaction at PCR cycles 14, 16, 18, and 20. Then add three microliters of 5X gel loading dye to each of the 12 microliter PCR aliquots, and to the 20 nucleotide ladder containing three microliters of ladder and nine microliters of RNase-free water. After running the samples on an agarose gel with 0.5 XTBE, evaluate the gel on a UV box to identify the optimal PCR cycle.Then set up the large-scale PCR reactions for agarose gel library purification. After PCR amplification, transfer nine microliters from each PCR tube into six 1.5 milliliter microcentrifuge tubes, containing three microliters of 5X gel loading dye. Prepare a 20 nucleotide ladder.Load the ladder, negative PCR control, and six PCR products onto the agarose gel, and run the gel for 30 minutes at 120 volts. Verify the uniformity of the PCR implifications between lanes on a UV box, and take an image. Combine three PCR reactions into two 1.5 milliliter siliconized microcentrifuge tubes, add 27 microliters of five molar sodium chloride, and 950 microliters of 100%ethanol.Invert the tubes to mix, and set them at 20 degrees celsius overnight to precipitate the PCR products. Using the protocol, the micro RNA expression correlation between the two fresh frozen tumor RNA and their specific FFPERNA specimen counterparts were ascertained. The plots between matched fresh-frozen and FFPE micro RNA indicate that the CDNA Library preparation provides a good reproducibility, as there is high correlation between the microRNA detected in specimens processed differently.To determine if the optimized CNDA Library preparation protocol was applicable and reproducible with increasingly older FFPE specimens, micro RNA expression profiles from FFPE tissues archived for 18, 20, 22, 27, 30, and 35 years were obtained. The correlation coefficient remained above 0.96 regardless of the age of the specimen or the library preparation week. Therefore, the optimized CDNA Library preparation protocol provides a robust tool for reproducible analysis of FFPE specimens, regardless of their archival time.So after watching this video you should have a good understanding on how to prepare your small RNA libraries and obtain a full microinexpression profile of all your precious formal and fixed paraffin-embedded specimens.

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Research

JoVE Journal

Genetics

Methyl-binding DNA capture Sequencing for Patient Tissues

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable development and differentiation of different tissue types. Dysregulation of this process is often the hallmark of various diseases like cancer. Here, we outline one of the recent sequencing techniques, Methyl-Binding DNA Capture sequencing (MBDCap-seq), used to quantify methylation in various normal and disease tissues for large patient cohorts. We describe a detailed protocol of this affinity enrichment approach along with a bioinformatics pipeline to achieve optimal quantification. This technique has been used to sequence hundreds of patients across various cancer types as a part of the 1,000 methylome project (Cancer Methylome System).

Here we present a protocol to investigate genome wide DNA methylation in large scale clinical patient screening studies using the Methyl-Binding DNA Capture sequencing (MBDCap-seq or MBD-seq) technology and the subsequent bioinformatics analysis pipeline.

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable ...

Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time PCR (RT-qPCR). It provides accurate, sensitive, reliable, and reproducible results. Several factors can affect the sensitivity and specificity of RT-qPCR. Residual genomic DNA (gDNA) contaminating RNA samples is one of them. In gene expression analysis, non-specific amplification due to gDNA contamination will overestimate the abundance of transcript levels and can affect the RT-qPCR results. Generally, gDNA is detected by qRT-PCR using primer pairs annealing to intergenic regions or an intron of the gene of interest. Unfortunately, intron/exon annotations are not yet known for all genes from vertebrate, bacteria, protist, fungi, plant, and invertebrate metazoan species.
Here we present a protocol for detection of gDNA contamination in RNA samples by using ribosomal DNA (rDNA)-based primers. The method is based on the unique features of rDNA: their multigene nature, highly conserved sequences, and high frequency in the genome. Also as a case study, a unique set of primers were designed based on the conserved region of ribosomal DNA (rDNA) in the Poaceae family. The universality of these primer pairs was tested by melt curve analysis and agarose gel electrophoresis. Although our method explains how rDNA-based primers can be applied for the gDNA contamination assay in the Poaceae family,&#160;it could be easily used to other prokaryote and eukaryote species

Here, we present a protocol for tracing genomic DNA (gDNA) contamination in RNA samples. The presented method utilizes primers specific for the internal transcribed spacer region (ITS) of ribosomal DNA (rDNA) genes. The method is suited for reliable and sensitive detection of DNA contamination in most eukaryotes and prokaryotes.

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time ...

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 nuclease or a nuclease-dead dCas9 fusion protein) is targeted to a specific site in the genome by a small synthetic RNA known as the guide RNA, or gRNA. The bipartite nature of the CRISPR system enables its use in screening approaches since plasmid libraries containing expression cassettes of thousands of individual gRNAs can be used to interrogate many different sites in a single experiment. 
To date, gRNA sequences for the construction of libraries have been almost exclusively generated by oligonucleotide synthesis, which limits the achievable complexity of sequences in the library and is relatively cost-intensive. Here, a detailed protocol for CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a simple and cost-effective method for the generation of highly complex gRNA libraries based on enzymatic digestion of input DNA, is described. Since CORALINA libraries can be generated from any source of DNA, plenty of options for customization exist, enabling a large variety of CRISPR-based screens.

Methods for generating large-scale gRNA libraries should be simple, efficient and cost-effective. We describe a protocol for the production of gRNA libraries based on enzymatic digestion of target DNA. This method, CORALINA (comprehensive gRNA library generation through controlled nuclease activity) presents an alternative to costly custom oligonucleotide synthesis.

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 ...

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

This article demonstrates a detailed protocol for DNA isolation and high-throughput sequencing library construction from herbarium material including rescue of exceptionally poor-quality DNA.

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been used to analyze the spectrum of clonal mutations in malignancy. Though far more efficient than traditional Sanger methods, NGS struggles with identifying rare clonal and subclonal mutations due to its high error rate of ~0.5&#8211;2.0%. Thus, standard NGS has a limit of detection for mutations that are &#62;0.02 variant allele fraction (VAF). While the clinical significance for mutations this rare in patients without known disease remains unclear, patients treated for leukemia have significantly improved outcomes when residual disease is &#60;0.0001 by flow cytometry. In order to mitigate this artefactual background of NGS, numerous methods have been developed. Here we describe a method for Error-corrected DNA and RNA Sequencing (ECS), which involves tagging individual molecules with both a 16 bp random index for error-correction and an 8 bp patient-specific index for multiplexing. Our method can detect and track clonal mutations at variant allele fractions (VAFs) two orders of magnitude lower than the detection limit of NGS and as rare as 0.0001 VAF.

Next-generation sequencing (NGS) is a powerful tool for genomic characterization that is limited by the high error rate of the platform (~0.5&#8211;2.0%). We describe our methods of error-corrected sequencing that allow us to obviate the NGS error rate and detect mutations at variant allele fractions as rare as 0.0001.

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state when water is supplied. The genomic sequencing of microscopic animals such as tardigrades risks bacterial contamination that sometimes leads to erroneous interpretations, for example, regarding the extent of horizontal gene transfer in these animals. Here, we provide an ultralow input method to sequence the genome of the tardigrade, Hypsibius dujardini, from a single specimen. By employing rigorous washing and contaminant exclusion along with an efficient extraction of the 50 ~ 200 pg genomic DNA from a single individual, we constructed a library sequenced with a DNA sequencing instrument. These libraries were highly reproducible and unbiased, and an informatics analysis of the sequenced reads with other H. dujardini genomes showed a minimal amount of contamination. This method can be applied to unculturable tardigrades that could not be sequenced using previous methods.

Contamination during the genomic sequencing of microscopic organisms remains a large problem. Here, we show a method to sequence the genome of a tardigrade from a single specimen with as little as 50 pg of genomic DNA without whole genome amplification to minimize the risk of contamination.

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state ...

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, and was deployed to West Africa during the recent Ebola virus epidemic, highlighting this possibility. In addition to its practical advantages (low cost, ease of equipment transport and use), this technology also provides fundamental advantages over second-generation sequencing approaches, particularly the very long read length, ability to directly sequence RNA, and real-time availability of data. Raw read accuracy is lower than with other sequencing platforms, which represents the main limitation of this technology; however, this can be partially mitigated by the high read depth generated. Here, we present a field-compatible protocol for sequencing of the mRNAs encoding for Niemann-Pick C1, which is the cellular receptor for ebolaviruses. This protocol encompasses extraction of RNA from animal blood samples, followed by RT-PCR for target enrichment, barcoding, library preparation, and the sequencing run itself, and can be easily adapted for use with other DNA or RNA targets.

Nanopore sequencing is a novel technology that allows cost-effective sequencing in remote locations and resource-poor settings. Here, we present a protocol for sequencing of mRNAs from whole blood that is compatible with such conditions.

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, ...

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT employs two purification steps. First, immunoprecipitation of a tagged RNP subunit is followed by mild RNase digestion and subsequent non-denaturing affinity elution. A second immunoprecipitation of another RNP subunit allows for enrichment of a defined complex. Following a denaturing elution of RNAs and proteins, the RNA footprints are converted into high-throughput DNA sequencing libraries. Unlike the more popular ultraviolet (UV) crosslinking followed by immunoprecipitation (CLIP) approach to enrich RBP binding sites, RIPiT is UV-crosslinking independent. Hence RIPiT can be applied to numerous proteins present in the RNA interactome and beyond that are essential to RNA regulation but do not directly contact the RNA or UV-crosslink poorly to RNA. The two purification steps in RIPiT provide an additional advantage of identifying binding sites where a protein of interest acts in partnership with another cofactor. The double purification strategy also serves to enhance signal by limiting background. Here, we provide a step-wise procedure to perform RIPiT and to generate high-throughput sequencing libraries from isolated RNA footprints. We also outline RIPiT&#39;s advantages and applications and discuss some of its limitations.

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing RIPiT-Seq

Here, we present a protocol to enrich endogenous RNA binding sites or &#34;footprints&#34;&#160;of RNA:protein (RNP) complexes from mammalian cells. This approach involves two immunoprecipitations of RNP subunits and is therefore dubbed RNA immunoprecipitation in tandem (RIPiT).

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT ...

High-Density DNA and RNA microarrays - Photolithographic Synthesis, Hybridization and Preparation of Large Nucleic Acid Libraries

Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite coupling reactions with the precision and density of UV light reflected from micrometer-sized mirrors. Photolithography yields microarrays that can accommodate from hundreds of thousands up to several million different DNA sequences, 100-nt or longer, in only a few hours. With this very large sequence space, microarrays are ideal platforms for exploring the mechanisms of nucleic acid&#183;ligand interactions, which are particularly relevant in the case of RNA. We recently reported on the preparation of a new set of RNA phosphoramidites compatible with in situ photolithography and which were subsequently used to grow RNA oligonucleotides, homopolymers as well as mixed-base sequences. Here, we illustrate in detail the process of RNA microarray fabrication, from the experimental design, to instrumental setup, array synthesis, deprotection and final hybridization assay using a template 25mer sequence containing all four bases as an example. In parallel, we go beyond hybridization-based experiments and exploit microarray photolithography as an inexpensive gateway to complex nucleic acid libraries. To do so, high-density DNA microarrays are fabricated on a base-sensitive monomer that allows the DNA to be conveniently cleaved and retrieved after synthesis and deprotection. The fabrication protocol is optimized so as to limit the number of synthetic errors and to that effect, a layer of &#946;-carotene solution is introduced to absorb UV photons that may otherwise reflect back onto the synthesis substrates. We describe in a step-by-step manner the complete process of library preparation, from design to cleavage and quantification.

In this article, we present and discuss new developments in the synthesis and applications of nucleic acid microarrays fabricated in situ. Specifically, we show how the protocols for DNA synthesis can be extended to RNA and how microarrays can be used to create retrievable nucleic acid libraries.

Photolithography is a powerful technique for the synthesis of DNA oligonucleotides on glass slides, as it combines the efficiency of phosphoramidite ...

Identification of Circular RNAs using RNA Sequencing

Circular RNAs (circRNAs) are a class of non-coding RNAs involved in functions including micro-RNA (miRNA) regulation, mediation of protein-protein interactions, and regulation of parental gene transcription. In classical next generation RNA sequencing (RNA-seq), circRNAs are typically overlooked as a result of poly-A selection during construction of mRNA libraries, or are found at very low abundance, and are therefore difficult to isolate and detect. Here, a circRNA library construction protocol was optimized by comparing library preparation kits, pre-treatment options and various total RNA input amounts. Two commercially available whole transcriptome library preparation kits, with and without RNase R pre-treatment, and using variable amounts of total RNA input (1 to 4 &#181;g), were tested. Lastly, multiple tissue types; including liver, lung, lymph node, and pancreas; as well as multiple brain regions; including the cerebellum, inferior parietal lobe, middle temporal gyrus, occipital cortex, and superior frontal gyrus; were compared to evaluate circRNA abundance across tissue types. Analysis of the generated RNA-seq data using six different circRNA detection tools (find_circ, CIRI, Mapsplice, KNIFE, DCC, and CIRCexplorer) revealed that a stranded total RNA library preparation kit with RNase R pre-treatment and 4 &#181;g RNA input is the optimal method for identifying the highest relative number of circRNAs. Consistent with previous findings, the highest enrichment of circRNAs was observed in brain tissues compared to other tissue types.

Circular RNAs (circRNAs) are non-coding RNAs that may have roles in transcriptional regulation and mediating interactions between proteins. Following assessment of different parameters for construction of circRNA sequencing libraries, a protocol was compiled utilizing stranded total RNA library preparation with RNase R pre-treatment and is presented here.