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. A., Ramnauth, A., Liu, C., Wang, T., Stead, 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=Hsa-miR-375+is+differentially+expressed+during+breast+lobular+neoplasia+and+promotes+loss+of+mammary+acinar+polarity.">Hsa-miR-375 is differentially expressed during breast lobular neoplasia and promotes loss of mammary acinar polarity.</a> J. Pathol. 226, 108-119 (2012).</li><li>Hui, A. B., Shi, W., Boutros, P. C., Miller, N., Pintilie, M., Fyles, T., 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=Robust+global+micro-RNA+profiling+with+formalin-fixed+paraffin-embedded+breast+cancer+tissues.">Robust global micro-RNA profiling with formalin-fixed paraffin-embedded breast cancer tissues.</a> Lab. Invest. 89, 597-606 (2009).</li><li>Zhang, X., Chen, J., Radcliffe, T., Lebrun, D. P., Tron, V. A., Feilotter, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+array-based+analysis+of+microRNA+expression+comparing+matched+frozen+and+formalin-fixed+paraffin-embedded+human+tissue+samples.">An array-based analysis of microRNA expression comparing matched frozen and formalin-fixed paraffin-embedded human tissue samples.</a> J. Mol. Diagn. 10, 513-519 (2008).</li><li>Kolbert, C. P., Feddersen, R. M., Rakhshan, F., Grill, D. E., Simon, G., 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=Multi-platform+analysis+of+microRNA+expression+measurements+in+RNA+from+fresh+frozen+and+FFPE+tissues.">Multi-platform analysis of microRNA expression measurements in RNA from fresh frozen and FFPE tissues.</a> PLoS One. 8, e52517 (2013).</li><li>Meng, W., McElroy, J. P., Volinia, S., Palatini, J., Warner, S., Ayers, L. W., 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=Comparison+of+microRNA+deep+sequencing+of+matched+formalin-fixed+paraffin-embedded+and+fresh+frozen+cancer+tissues.">Comparison of microRNA deep sequencing of matched formalin-fixed paraffin-embedded and fresh frozen cancer tissues.</a> PLoS One. 8 (5), e64393 (2013).</li><li>Liu, A., Tetzlaff, M. T., Vanbelle, P., Elder, D., Feldman, M., Tobias, J. W., 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+profiling+outperforms+mRNA+expression+profiling+in+formalin-fixed+paraffin-embedded+tissues.">MicroRNA expression profiling outperforms mRNA expression profiling in formalin-fixed paraffin-embedded tissues.</a> Int. J. Clin. Exp. Pathol. 2, 519-527 (2009).</li><li>Dijkstra, J. R., Mekenkamp, L. J., Teerenstra, S., De Krijger, I., Nagtegaal, I. D. <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+in+formalin-fixed+paraffin+embedded+tissue+using+real+time+quantitative+PCR:+the+strengths+and+pitfalls.">MicroRNA expression in formalin-fixed paraffin embedded tissue using real time quantitative PCR: the strengths and pitfalls.</a> J Cell Mol Med. 16, 683-690 (2012).</li><li>Siebolts, U., Varnholt, H., Drebber, U., Dienes, H. P., Wickenhauser, C., Odenthal, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissues+from+routine+pathology+archives+are+suitable+for+microRNA+analyses+by+quantitative+PCR.">Tissues from routine pathology archives are suitable for microRNA analyses by quantitative PCR.</a> J. Clin. 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. E., Rubio, R., Duan, S., 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=Next-generation+sequencing+and+microarray-based+interrogation+of+microRNAs+from+formalin-fixed,+paraffin-embedded+tissue:+preliminary+assessment+of+cross-platform+concordance.">Next-generation sequencing and microarray-based interrogation of microRNAs from formalin-fixed, paraffin-embedded tissue: preliminary assessment of cross-platform concordance.</a> Genomics. 102, 8-14 (2013).</li><li>Visone, R., Croce, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MiRNAs+and+cancer.">MiRNAs and cancer.</a> Am. J. Pathol. 174, 1131-1138 (2009).</li><li>Lu, J., Getz, G., Miska, E. 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. S., Lin, J., Isayeva, T., Liu, C., 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=Illumina+whole-genome+complementary+DNA-mediated+annealing,+selection,+extension+and+ligation+platform:+assessing+its+performance+in+formalin-fixed,+paraffin-embedded+samples+and+identifying+invasion+pattern-related+genes+in+oral+squamous+cell+carcinoma.">Illumina whole-genome complementary DNA-mediated annealing, selection, extension and ligation platform: assessing its performance in formalin-fixed, paraffin-embedded samples and identifying invasion pattern-related genes in oral squamous cell carcinoma.</a> Hum. Pathol. 42 (12), 1911-1922 (2011).</li></ol>

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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Genetics

9.2K vistas.  Hackensack University Medical Center. 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 ...