Name: retrospective microrna sequencing: complementary dna library preparation protocol using formalin-fixed paraffin-embedded rna specimens
Uploaded: 2018-05-05T13:00:00.000+00:00
Duration: 12 min 24 s
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.

<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>

The authors wish to disclose that a publication containing some of the data presented in this manuscript was published in the International Journal of Molecular Sciences by Loudig et al.21.

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 pooling of all RNA samples (i.e., 18 individual 100 ng FFPE RNA samples) after individual ligations with the 18 unique 3&#39; barcoded adapters. This critical step allows for the 18 FFPE RNA samples to uniformly undergo all subsequent biochemical and enzymatic steps necessary for preparation of the small RNA cDNA library. Additionally, this step maximizes the amount of RNA undergoing precipitations and gel purifications by enhancing the carrier effect of the small RNA molecules and by facilitating pellet observation and gel selections. Another key feature of this protocol, which further highlights its versatility when working with small amounts of FFPE RNA, is that a pilot PCR reaction is used to identify the optimal amplification cycle of the cDNA library. This step also provides insights into the dynamics of the library amplification versus primer dimer amplification, which is critical when preparing the large-scale PCR reaction and purifying the small RNA library on the agarose gel. The data added to this protocol demonstrate the reproducibility of this approach between matched frozen and FFPE specimens, and also highlight its applicability to older FFPE RNA samples from specimens archived for up to 35 years.
This protocol was modified from its original version, so it is optimized for preparation of small RNA libraries made from lower quality and quantity of chemically modified and compromised FFPE RNA. One aspect of this procedure that was modified to successfully ligate and amplify small RNAs from FFPE specimens relates to the amount of calibrator cocktail spiked during the initial 3&#39; barcoded adapter ligation, for which input was reduced to 0.026 nM to prevent competition with the ligation of low abundancy small RNAs present in FFPE RNA samples. Another important modification to the original procedure was the removal of all radioactively labeled size-markers used during selection of the ligated small RNAs on the PAGE gels. Use of these radiolabeled size-markers not only restricted the applicability of this approach to laboratories certified for use of radio-isotopes but also prevented observation of the RNA products on the gels. Instead, in this optimized protocol, size markers are run in wells adjacent to the library, on the PAGE gel, and visualized directly with a fluorescent dye to direct excision of the ligated small RNAs. Importantly, the fluorescent dye is lightly sprayed on the gel to prevent diffusion of the RNA products and then visualized on a blue light box. Subsequently, as a measure of precaution and to improve the diffusion of the ligated small RNAs contained in the excised PAGE gel fragments, gel-breaker tubes are used to uniformly crush the gel prior to incubation in a NaCl solution overnight at 4 &#176;C under agitation. All these steps were carefully evaluated to work with smaller amounts of material.
This protocol was applied to FFPE RNA samples from different sources (organs and institutions) that had been stored for different amounts of time. The analyses revealed the high reproducibility of sequencing small RNAs from fresh and frozen specimens, when using this optimized procedure. Additional analyses using older archived FFPE specimens, stored for up to 35 years, further demonstrated the protocol&#39;s vast applicability to clinical specimens. The minimum FFPE RNA input requirement for FFPE specimens was shown to be 100 ng. This low requirement allows applicability of this protocol to a variety of lesions of different sizes and availability, but it would not allow the analysis of single cell FFPE RNA. It is important to note, however, that this optimized protocol has also been used with total RNA extracted from circulating exosomes with inputs of less than 1 ng and shown to provide highly reproducible small RNA expression profiles (data not shown). This low input suggests that small RNAs in FFPE samples, although representative of the original fresh tissue, are present in much lower proportions than in total RNA from circulating exosomes.
In recent work, where this optimized protocol was applied to clinically classified DCIS FFPE specimens, it was found that miRNA expression differences identified by NGS of the cDNA library could be validated by quantitative PCR. This work demonstrated the feasibility of using DCIS lesions from archived tissues from different institutions for large-scale retrospective studies [20]. This study also highlighted the robustness of this cDNA library preparation when performed at different times (at intervals of several weeks), without compromising reproducibility and sensitivity of the assay.
Considering the vast amount of clinically classified archived FFPE specimens, this optimized protocol provides a robust tool for preparation of cDNA libraries in large-scale retrospective analyses and for the potential identification of miRNA biomarkers associated with cancer development21.

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&amp;trade; 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 of the PAGE gel, are used to select the appropriately sized 3&#39; barcoded small RNAs (Figure 2). The excised gel piece is incubated in a NaCl solution overnight to elute the ligated RNA molecules. The next day, the eluted RNA is precipitated, and a 5&#39; adapter ligation is performed. Then, the 5&#39; adapter ligated small RNA molecules are migrated and separated on a 12% acrylamide gel, where again migrated RNA size marker oligonucleotides allow size excision of the small RNAs containing both the 3&#39; barcoded oligonucleotides and the 5&#39; adapter (Figure 3). The excised gel is incubated overnight in a NaCl solution to allow elution of the RNA. The next day, the ligated small RNA molecules are precipitated, and the pellet is resuspended in RNase-free water, followed by reverse-transcription; an aliquot of the cDNA molecules undergoes a pilot PCR reaction (Figure 4A). Large-scale PCR reactions using the same input of cDNA libraries are set up and evaluated on a 2.5% agarose gel to verify that all reactions were adequately PCR amplified (Figure 4B), prior to pooling and overnight ethanol precipitation. The next day, the amplified cDNA library containing all 18 individual libraries for the 18 unique FFPE RNA specimens, is migrated on a 2.5% agarose gel, and the top PCR band, running at 100 nt is excised and purified (Figure 4C). The cDNA library purification is then evaluated on a high sensitivity DNA chip (Figure 5) to determine that the purified PCR product does not contain an excess of primer dimers or other byproducts of the PCR reaction. The PCR product is then analyzed on a high-throughput sequencing system. The adapter trimming and generation of 18 individual files for each of the 18 specimens are performed using the RNAworld pipeline (access was provided to us by Dr. Thomas Tuschl). Biostatistical analyses are then performed to evaluate the miRNA contents of the FFPE RNA specimens.
To validate this optimized procedure, matched fresh frozen and FFPE breast tumor specimens were used for the analyses (Figure 6). Two similar invasive ductal breast carcinoma (IDC) tumors were selected to evaluate the sensitivity of the procedure and determine if miRNA expression differences identified between the two fresh frozen tissues could also be detected in the matched archived FFPE RNA specimens. For this experiment, the quality of the total RNA obtained from the 2 fresh frozen and the matched two FFPE RNA samples was evaluated (Figure 6A). As anticipated, the RNA size and the quality of the FFPE specimens was severely decreased when compared to the matched fresh frozen RNAs (compare lanes 1 and 3, and lanes 2 and 4). One of the FFPE RNA had been archived at RT for 4 years (Invasive Breast Cancer 1, (IBC1)) and the other had been archived at RT for 8 years (IBC2), while the fresh frozen counterparts had been stored at -80 &#176;C. The four individual RNA specimens were analyzed in a single library, using 4 individual barcodes, and the miRNA read distribution plots are displayed in Figure 6B. The two top panels display miRNA expression correlation between the two-fresh frozen tumor RNAs and their specific FFPE RNA specimen counterparts. The plots between matched fresh frozen and FFPE miRNAs indicate that the cDNA library preparation provides a good reproducibility as a high correlation can be observed between the miRNAs detected in specimens processed differently (Frozen vs. FFPE). The two lower panels display the correlation between miRNA expression data from the two different frozen tumors and between the two different FFPE tumors. As indicated in Figure 6C, the miRNA expression differences identified between the two fresh frozen tumors were correlated with the differences detected between the two matched FFPE tumor specimens. Significance miRNA expression differences were detectable both in fresh frozen and FFPE tumors.
To further evaluate the sensitivity of this approach, miRNA expression data from 12 archived FFPE specimens and 4 fresh frozen RNA specimens were used (Figure 6D). The 16 different RNA specimens were individually 3&#39; barcoded and all used for the preparation of a single cDNA library. The RNA samples used in this library included two breast cell lines, the MCF10A (normal-like cell line) and the MCF7 (breast cancer cell line) with RNA from fresh cells and from their archived FFPE counterparts23, matched fresh frozen and FFPE RNA samples from the two breast cancer samples analyzed independently (IBC1 and IBC2 in Figure 6A and 6B), and matched fresh frozen and FFPE RNA samples from normal cervical samples (Cx). Additionally, archived FFPE specimens from normal (normal Br1, normal Br2, and normal Br3), and cancer breast tissues (IBC 5, IBC 6, and IBC 7), without their fresh frozen counterparts were analyzed. As observed on the heatmap, regardless of fresh frozen or FFPE RNA origin, miRNA expression profiles of the same cells (MCF10 or MCF7), or the same tissues (IBC1, IBC2, or Cx) clustered together. Additionally, as noted on the unsupervised cluster, from left to right, normal breast cells and tissues clustered together while breast tumors and tumor cells clustered on the right. The cervical tissue, which displayed a different miRNA expression profile, clustered on the right of the heatmap.
Considering that most of the clinically archived FFPE specimens do not have fresh frozen counterparts but that they can be retrieved after different storage duration, it was sought to determine if the optimized cDNA library preparation protocol was applicable and reproducible with increasingly older FFPE specimens. As displayed in Figure 7, miRNA expression profiles from FFPE tissues archived for 18, 20, 22, 27, 30, and 35 years were obtained. The RNA was extracted using the optimized simultaneous RNA/DNA procedure21, and quadruplet RNA aliquots from each individual FFPE specimen were prepared on the same day and stored at -80 &#176;C prior to library preparations. A total of 9 different FFPE specimens were analyzed in duplicate, where each individual RNA aliquot was ligated with different barcoded oligonucleotides (18 barcodes total), within the same library. This experiment was repeated during two consecutive weeks (week 1 and week 2). This allowed evaluation of the cDNA library preparation reproducibility with the same RNA specimens using two different barcodes within a single library, and between two different libraries with a one-week interval. As observed on Figure 7, 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, for example, the 35-year-old archived FFPE RNA (see Breast #9) displayed high reproducible measures equivalent to those noted with the 20-year-old FFPE RNA samples (see Breast #3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57471/57471fig1.jpg" /> 
Figure 1: Oligonucleotides. All oligonucleotide sequences, their corresponding chemical modifications, and concentrations used in this protocol are described. The types of chemical modifications are described in the modification box and the abbreviated modifications displayed in the oligonucleotide sequences. The calibrator cocktail displays the list of the 10 individual RNA oligonucleotide calibrators, which were resuspended in a solution containing the carrier oligonucleotide (0.5 &#181;M). The eighteen 3&#39; barcoded oligonucleotide adapters are detailed with grey shading over the barcode&#39;s sequence. The RNA sequence of the 5&#39; adapter, and the DNA sequences of the 3&#39; PCR and 5&#39; PCR primers are detailed. The RNA sequences of the two size marker oligonucleotides, namely referenced in the protocol as 19 nt-3&#39; adapter and 24 nt-3&#39; adapter size markers, are provided. All DNA and RNA oligonucleotides were commercially purchased. <a href="/files/ftp_upload/57471/57471fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57471/57471fig2.jpg" /> 
Figure 2: PAGE purification of the 3&#39; barcoded RNA samples. After pooling and precipitating the 18 barcoded RNA samples, the resuspended RNA pellet is migrated and separated by electrophoresis on a 15% acrylamide gel (see well 8). The red square highlights the area containing the 3&#39; barcoded microRNAs, which was excised with a scalpel blade and transferred into a nuclease-free siliconized microcentrifuge tube. A total of four wells, with two containing the 19 nt-3&#39; adapter and two containing the 24 nt-3&#39; adapter was set a well away, on each side of the library (see wells 5, 6, and 10, 11, respectively). As a test for the T4 RNA ligase 1 and for purification of the ligated size markers to be completed the next day, ligation reactions containing the 19 nt-3&#39; adapter size marker with the 5&#39; adapter and the 24 nt-3&#39; adapter size marker with the 5&#39; adapter were run in wells 2 and 3, respectively. The yellow squares display the excised bands representing the ligated size marker RNA oligonucleotides with the 5&#39; adapter. These bands are purified, precipitated, and run during on the 12% PAGE purification (see Figure 3 below). Wells 1 and 13 contain the 20 nt size ladder, which helped confirm the anticipated sizes of the ligated products. <a href="/files/ftp_upload/57471/57471fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57471/57471fig3.jpg" /> 
Figure 3: PAGE purification of the purified library after ligation of the 5&#39; adapter. The purified RNA library was ligated with the 5&#39; adapter and the expected product size was excised from the 12% PAGE using the ligated size markers (see red square). The products of the T4 RNA ligations between the 19 nt-3&#39; adapter and the 5&#39; adapter (wells 4 and 9) and between the 24 nt-3&#39; adapter and the 5&#39; adapter (wells 5 and 10) were run in parallel, on each side of the well containing the RNA library. The highest bands (see white asterisks) are the products of the 5&#39; adapter ligation and used as the guide for the gel band excision containing the 5&#39; adapter ligated RNA library. The ligation size marker ligation products purified on the previous PAGE are run in well 3. As observed in wells 1 and 12, the 20 nt size ladder was also run to validate the anticipated size of the RNA library. <a href="/files/ftp_upload/57471/57471fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57471/57471fig4.jpg" /> 
Figure 4: Pilot PCR and large-scale amplification of the cDNA library. The size and ratio of the PCR products were evaluated on a 2.5% agarose gel in the presence of ethidium bromide. (A) After reverse transcription of the barcoded RNA library, an aliquot of the cDNA library was amplified using the 5&#39; and 3&#39; PCR primers in a single pilot PCR reaction. Aliquots of the pilot PCR reactions were obtained at 10, 12, 14, 16, 18, and 20 cycles and migrated on a 2.5% agarose gel. As observed between wells 1 and 7, the presence of the cDNA library and adapter dimers was exponentially observable (see green rectangles). For this library, the PCR cycle selected for amplification of the cDNA library was 15. (B) This schematic displays the position and length of the different oligonucleotides and the resulting RNA and DNA products, which are identifiable on the PAGE and agarose gels. (C) Aliquots of the 6 large-scale PCR reactions (identified in A) were analyzed separately on a 2.5% agarose gel (see wells 3 to 8). The two types of PCR products, namely the library (upper band) and the primers dimers (lower band) are visible on this gel (see green rectangles). The blank PCR reaction without cDNA displays no PCR amplification (see well 2). The 20 nt size ladder allows verification of the product sizes (see well 1). (D) Gel image on a blue light transilluminator of the 2.5% agarose gel containing the pooled PCR reactions ran in two adjacent wells. As observed, the highest band in both wells is within the expected library size and the adapter dimer band is located below. The upper PCR bands were excised and purified with a gel extraction kit. <a href="/files/ftp_upload/57471/57471fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57471/57471fig5.jpg" /> 
Figure 5: Evaluation of the purified PCR amplified DNA library. A small aliquot (1 &#181;L) of the PCR amplified cDNA library is analyzed using a high sensitivity DNA chip on a microfluidics-based platform. (A) This panel displays the migration of the size markers for calibration of the instrument. (B) This panel displays the migration of the purified PCR amplified cDNA library. The highest peak is measured at a size of 100 bp (see asterisk) and represents the cDNA library. The small peak evaluated at 72 bp by the instrument represents the primer adapter dimers. The 100 bp peak detected by microfluidics based platform reveals the estimated size for the amplified cDNA library, which contains 18 individual barcoded RNA specimens for subsequent NGS on a high-throughput sequencing system. <a href="/files/ftp_upload/57471/57471fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57471/57471fig6.jpg" /> 
Figure 6: cDNA library preparation and next-generation sequencing using matched fresh frozen and formalin-fixed paraffin embedded specimens. (A) Total RNA extracted from matched fresh frozen and FFPE invasive ductal carcinoma (IDC) tumors was analyzed on a total RNA chip on a microfluidics based platform. (B) Total RNA from matched fresh frozen and FFPE specimens (IBC 1 and IBC2) underwent the cDNA library preparation protocol and the miRNA sequencing data were plotted. (C) DifferentialmiRNA expression between IBC1/IBC2 specimens and correlation between frozen and FFPE paired samples. miRNA expression is displayed in log count per million (CPM), from high to low reads (red to blue color). The significance of the expression difference between the two tissue pairs, per miRNA, is displayed by the gray circles, with high and low expression miRNAs identified in fresh and FFPE samples. (D) Total RNA extracted from matched fresh and FFPE RNA specimens of breast cell lines (MCF10A and MCF7), human invasive breast cancer (IBC 1 and IBC2), cervical tissue (Cx), archived normal breast tissues (normal Br1, normal Br2, and normal Br 3), and archived invasive breast cancer (IBC 5, IBC 6, and IBC7) underwent the cDNA library preparation within the same run. The NGS data of the miRNAs detected in the libraries are displayed in a heat-map configuration. This figure has been modified from Loudig et al.20 <a href="/files/ftp_upload/57471/57471fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57471/57471fig7.jpg" /> 
Figure 7: cDNA library preparation and miRNA expression correlation between replicates using older archived FFPE specimens. Total RNA from 18, 20, 22, 27, 30, and 35-year-old archived FFPE breast tissue specimens underwent our optimized cDNA library preparation protocol. Replicate RNA samples from 9 different FFPE specimens were ligated with different 3&#39; barcoded oligonucleotides and analyzed within the same library on week 1 (w1). The same experiment was repeated within a one-week interval (week 2 or w2). Reproducibility measures of duplicated miRNA expression data between the same archived RNA specimens are displayed in the heatmaps and with correlation coefficients evaluated between 0.93 and 0.99. This figure has been modified from Loudig et al.20 <a href="/files/ftp_upload/57471/57471fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

retrospective-microrna-sequencing-complementary-dna-library

Nanyang Technological University

Research

JoVE Journal

Genetics

9.3K Views.  Hackensack University Medical Center. Paraffine-ingebedde specimens formaline-vaste vormen een waardevolle bron van moleculaire biomarkers voor ziekten bij de mens. Hier presenteren we een laboratorium gebaseerde cDNA Bibliotheek voorbereiding protocol, in eerste instantie ontworpen met vers bevroren RNA, en geoptimaliseerd voor de analyse van gearchiveerde microRNAs uit weefsels tot 35 jaar opgeslagen.

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

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, milk and urine. One subset of these RNAs are the posttranscriptional regulators &#8211; microRNAs (miRNAs). To delineate the miRNAs produced by specific cell types, in vitro culture systems can be used to harvest and profile exRNAs derived from one subset of cells. The secreted factors of mesenchymal stem cells are implicated in alleviating numerous diseases and is used as the in vitro model system here. This paper describes the process of collection, purification of small RNA and library generation to sequence extracellular miRNAs. ExRNAs from culture media differ from cellular RNA by being low RNA input samples, which calls for optimized procedures. This protocol provides a comprehensive guide to small exRNA sequencing from culture media, showing quality control checkpoints at each step during exRNA purification and sequencing.

This protocol demonstrates how to purify extracellular microRNAs from cell culture media for small RNA library construction and next generation sequencing. Various quality control checkpoints are described to allow readers to understand what to expect when working with low input samples like exRNAs.

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, ...

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 ...

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.

Circular RNAs (circRNAs) are a class of non-coding RNAs involved in functions including micro-RNA (miRNA) regulation, mediation of protein-protein ...

Sequencing Small Non-coding RNA from Formalin-fixed Tissues and Serum-derived Exosomes from Castration-resistant Prostate Cancer Patients

Ablation of androgen receptor (AR) signaling by androgen deprivation is the goal of the first line of therapy for prostate cancer that initially results in cancer regression. However, in a significant number of cases, the disease progresses to advanced, castration-resistant prostate cancer (CRPC), which has limited therapeutic options and is often aggressive. Distant metastasis is mostly observed at this stage of the aggressive disease. CRPC is treated by a second generation of AR pathway inhibitors that improve survival initially, followed by the emergence of therapy resistance. Neuroendocrine prostate cancer (NEPC) is a rare variant of prostate cancer (PCa) that often develops as a result of therapy resistance via a transdifferentiation process known as neuroendocrine differentiation (NED), wherein PCa cells undergo a lineage switch from adenocarcinomas and show increased expression of neuroendocrine (NE) lineage markers. In addition to the genomic alterations that drive the progression and transdifferentiation to NEPC, epigenetic factors and microenvironmental cues are considered essential players in driving disease progression. This manuscript provides a detailed protocol to identify the epigenetic drivers (i.e., small non-coding RNAs) that are associated with advanced PCa. Using purified microRNAs from formalin-fixed paraffin-embedded (FFPE) metastatic tissues and corresponding serum-derived extracellular vesicles (EVs), the protocol describes how to prepare libraries with appropriate quality control for sequencing microRNAs from these sample sources. Isolating RNA from both FFPE and EVs is often challenging because most of it is either degraded or is limited in quantity. This protocol will elaborate on different methods to optimize the RNA inputs and cDNA libraries to yield most specific reads and high-quality data upon sequencing.

Therapy resistance often develops in patients with advanced prostate cancer, and in some cases, cancer progresses to a lethal subtype called neuroendocrine prostate cancer. Assessing the small non-coding RNA-mediated molecular changes that facilitate this transition would allow better disease stratification and identification of causal mechanisms that lead to development of neuroendocrine prostate cancer.

Ablation of androgen receptor (AR) signaling by androgen deprivation is the goal of the first line of therapy for prostate cancer that initially results ...

Cancer Research

Optimization for Sequencing and Analysis of Degraded FFPE-RNA Samples

Gene expression analysis by RNA sequencing (RNA-seq) enables unique insights into clinical samples that can potentially lead to mechanistic understanding of the basis of various diseases as well as resistance and/or susceptibility mechanisms. However, FFPE tissues, which represent the most common method for preserving tissue morphology in clinical specimens, are not the best sources for gene expression profiling analysis. The RNA obtained from such samples is often degraded, fragmented, and chemically modified, which leads to suboptimal sequencing libraries. In turn, these generate poor quality sequence data that may not be reliable for gene expression analysis and mutation discovery. In order to make the most of FFPE samples and obtain the best possible data from low quality samples, it is important to take certain precautions while planning experimental design, preparing sequencing libraries, and during data analysis. This includes the use of appropriate metrics for precise sample quality control (QC), identifying the best methods for various steps during the sequencing library generation, and careful library QC. In addition, applying correct software tools and parameters for sequence data analysis is critical in order to identify artifacts in RNA-seq data, filter out contamination and low quality reads, assess uniformity of gene coverage, and measure the reproducibility of gene expression profiles among biological replicates. These steps can ensure high accuracy and reproducibility for profiling of very heterogeneous RNA samples. Here we describe the various steps for sample QC, library preparation and QC, sequencing, and data analysis that can help to increase the amount of useful data obtained from low quality RNA, such as that obtained from FFPE-RNA tissues.

This method describes the steps to improve the quality and quantity of sequence data that can be obtained from formalin-fixed paraffin-embedded (FFPE) RNA samples. We describe the methodology to more accurately assess the quality of FFPE-RNA samples, prepare sequencing libraries, and analyze the data from FFPE-RNA samples.

Gene expression analysis by RNA sequencing (RNA-seq) enables unique insights into clinical samples that can potentially lead to mechanistic understanding ...

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

Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other genome-wide analyses. mRNA-Seq even opens the gate for gene expression analysis of non-sequenced genomes. mRNA-Seq offers high sensitivity, a large dynamic range and allows measurement of transcript copy numbers in a sample. Illumina&#x2019;s genome analyzer performs sequencing of a large number (&gt; 107) of relatively short sequence reads (&lt; 150 bp).The &quot;paired end&quot; approach, wherein a single long read is sequenced at both its ends, allows for tracking alternate splice junctions, insertions and deletions, and is useful for de novo transcriptome assembly.
One of the major challenges faced by researchers is a limited amount of starting material. For example, in experiments where cells are harvested by laser micro-dissection, available starting total RNA may measure in nanograms. Preparation of mRNA-Seq libraries from such samples have been described1, 2 but involves significant PCR amplification that may introduce bias. Other RNA-Seq library construction procedures with minimal PCR amplification have been published3, 4 but require microgram amounts of starting total RNA.
Here we describe a protocol for the Illumina Genome Analyzer II platform for mRNA-Seq sequencing for library preparation that avoids significant PCR amplification and requires only 10 nanograms of total RNA. While this protocol has been described previously and validated for single-end sequencing5, where it was shown to produce directional libraries without introducing significant amplification bias, here we validate it further for use as a paired end protocol. We selectively amplify polyadenylated messenger RNAs from starting total RNA using the T7 based Eberwine linear amplification method, coined &quot;T7LA&quot; (T7 linear amplification). The amplified poly-A mRNAs are fragmented, reverse transcribed and adapter ligated to produce the final sequencing library. For both single read and paired end runs, sequences are mapped to the human transcriptome6 and normalized so that data from multiple runs can be compared. We report the gene expression measurement in units of transcripts per million (TPM), which is a superior measure to RPKM when comparing samples7.

Here we describe a method for preparation of both single read and paired end Illumina mRNA-Seq sequencing libraries for gene expression analysis based on T7 linear RNA amplification. This protocol requires only 10 nanograms of starting total RNA and generates highly consistent libraries representing whole transcripts.

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other ...

Biology

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.

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 ...

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding RNAs. Standard RNAseq requires 30 - 100 million sequencing reads and can include multiple RNA products such as mRNA and noncoding RNAs. We demonstrate how targeted RNAseq (capture) permits a focused study on selected RNA products using a desktop sequencer. RNAseq capture can characterize unannotated, low, or transiently expressed transcripts that may otherwise be missed using traditional RNAseq methods. Here we describe the extraction of RNA from cell lines, ribosomal RNA depletion, cDNA synthesis, preparation of barcoded libraries, hybridization and capture of targeted transcripts and multiplex sequencing on a desktop sequencer. We also outline the computational analysis pipeline, which includes quality control assessment, alignment, fusion detection, gene expression quantification and identification of single nucleotide variants. This assay allows for targeted transcript sequencing to characterize gene expression, gene fusions, and mutations.

We describe a targeted RNA sequencing-based method that includes preparation of indexed cDNA libraries, hybridization and capture with custom probes and data analysis to interrogate selected transcripts for gene expression, mutations, and gene fusions. Targeted RNAseq permits cost-effective, rapid evaluation of selected transcripts on a desktop sequencer.

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding ...

Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

This modified extraction protocol improves RNA and DNA yields from more precisely targeted regions of interest in histopathologic tissue blocks.

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in ...

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

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