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>
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Pathol. 62, 84-88 (2009).</li><li>Jang, J. S., Simon, V. A., Feddersen, R. M., Rakhshan, F., Schultz, D. A., Zschunke, M. A., Lingle, W. L., Kolbert, C. P., Jen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+miRNA+expression+analysis+using+fluidigm+microfluidics+dynamic+arrays.">Quantitative miRNA expression analysis using fluidigm microfluidics dynamic arrays.</a> BMC Genomics. 12, 144 (2011).</li><li>Andreasen, D., Fog, J. U., Biggs, W., Salomon, J., Dahslveen, I. K., Baker, A., Mouritzen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+microRNA+quantification+in+total+RNA+from+clinical+samples.">Improved microRNA quantification in total RNA from clinical samples.</a> Methods. 50 (4), S6-S9 (2010).</li><li>Kelly, A. D., Hill, K. E., Correll, M., Wang, Y. 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A., Alvarez-Saavedra, E., Lamb, J., Peck, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+expression+profiles+classify+human+cancers.">MicroRNA expression profiles classify human cancers.</a> Nature. 435, 834-838 (2005).</li><li>Cho, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs:+potential+biomarkers+for+cancer+diagnosis,+prognosis+and+targets+for+therapy.">MicroRNAs: potential biomarkers for cancer diagnosis, prognosis and targets for therapy.</a> Int. J. Biochem. Cell. Biol. 42, 1273-1281 (2010).</li><li>Hui, A., How, C., Ito, E., Liu, F. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-RNAs+as+diagnostic+or+prognostic+markers+in+human+epithelial+malignancies.">Micro-RNAs as diagnostic or prognostic markers in human epithelial malignancies.</a> BMC Cancer. 11, 500 (2011).</li><li>Tam, S., de Borja, R., Tsao, M. S., McPherson, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+global+microRNA+expression+profiling+using+next-generation+sequencing+technologies.">Robust global microRNA expression profiling using next-generation sequencing technologies.</a> Lab. Invest. 94, 350-358 (2014).</li><li>Loudig, O., Wan, T., Ye, K., Lin, J., Wang, Y., Ramnauth, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+and+Adaptation+of+a+Laboratory+Based+cDNA+Library+Preparation+Protocol+for+Retrospective+Sequencing+of+Archived+MicroRNAs+from+up+to+35-Year-Old+Clinical+FFPE+Specimens.">Evaluation and Adaptation of a Laboratory Based cDNA Library Preparation Protocol for Retrospective Sequencing of Archived MicroRNAs from up to 35-Year-Old Clinical FFPE Specimens.</a> Int J Mol Sci. 18, e627 (2017).</li><li>Kotorashvili, A., Ramnauth, A., Liu, C., Lin, J., Ye, K., Kim, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+DNA/RNA+co-extraction+for+analysis+of+microRNAs,+mRNAs,+and+genomic+DNA+from+formalin-fixed+paraffin-embedded+specimens.">Effective DNA/RNA co-extraction for analysis of microRNAs, mRNAs, and genomic DNA from formalin-fixed paraffin-embedded specimens.</a> PLoS One. 7 (4), e34683 (2012).</li><li>Hafner, M., Renwick, N., Farazi, T. A., Mihailovi&#263;, A., Pena, J. T., Tuschl, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barcoded+cDNA+library+preparation+for+small+RNA+profiling+by+next-generation+sequencing.">Barcoded cDNA library preparation for small RNA profiling by next-generation sequencing.</a> Methods. 58 (2), 164-170 (2012).</li><li>Loudig, O., Brandwein-Gensler, M., Kim, R. 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&#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 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. 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.

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

Methyl-binding DNA capture Sequencing for Patient Tissues

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

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

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

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA interactions such as microRNA-mRNA interactions have been shown to regulate gene expression, the full extent to which RNA interactions occur in the cell is still unknown. Previous methods to study RNA interactions have primarily focused on subsets of RNAs that are interacting with a particular protein or RNA species. Here, we detail a method named Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH) that allows genome-wide capture of RNA interactions in vivo in an unbiased manner. SPLASH utilizes in vivo crosslinking, proximity ligation, and high throughput sequencing to identify intramolecular and intermolecular RNA base-pairing partners globally. SPLASH can be applied to different organisms including bacteria, yeast and human cells, as well as diverse cellular conditions to facilitate the understanding of the dynamics of RNA organization under diverse cellular contexts. The entire experimental SPLASH protocol takes about 5 days to complete and the computational workflow takes about 7 days to complete.

Here, we detail the method of Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH), which enables genome-wide mapping of intramolecular and intermolecular RNA-RNA interactions in vivo. SPLASH can be applied to study RNA interactomes of organisms including yeast, bacteria and humans.

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA

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

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

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

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

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

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

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

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

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

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

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

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

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

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

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

Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen

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

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

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

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

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

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

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

An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including cancers. These small regulatory RNAs mainly interact with the 3&#8217; untranslated regions (3&#8217; UTR) of their target messenger RNAs (mRNAs) ultimately resulting in the inhibition of decoding processes of mRNAs and the augmentation of target mRNA degradations. Based on the expression levels and intracellular functions, miRNAs are able to serve as regulatory factors of oncogenic and tumor-suppressive mRNAs. Identification of bona fide target genes of a miRNA among hundreds or even thousands of computationally predicted targets is a crucial step to discern the roles and basic molecular mechanisms of a miRNA of interest. Various miRNA target prediction programs are available to search possible miRNA-mRNA interactions. However, the most challenging question is how to validate direct target genes of a miRNA of interest. This protocol describes a reproducible strategy of key methods on how to identify miRNA targets related to the function of a miRNA. This protocol presents a practical guide on step-by-step procedures to uncover miRNA levels, functions, and related target mRNAs using the probe-based real-time polymerase chain reaction (PCR), sulforhodamine B (SRB) assay following a miRNA mimic transfection, dose-response curve generation, and luciferase assay along with the cloning of 3&#8217; UTR of a gene, which is necessary for proper understanding of the roles of individual miRNAs.

This protocol uses a probe-based real-time polymerase chain reaction (PCR), a sulforhodamine B (SRB) assay, 3&#8217; untranslated regions (3&#8217; UTR) cloning, and a luciferase assay to verify the target genes of a miRNA of interest and to understand the functions of miRNAs.

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including ...

Single-cell RNA Sequencing and Analysis of Human Pancreatic Islets

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to normal and pathological conditions. The goal of this protocol is to generate high-quality, large-scale transcriptome data of each endocrine cell type with the use of a droplet-based microfluidic single-cell RNA sequencing technology. Such data can be utilized to build the gene expression profile of each endocrine cell type in normal or specific conditions. The process requires careful handling, accurate measurement, and rigorous quality control. In this protocol, we describe detailed steps for human pancreatic islets dissociation, sequencing, and data analysis. The representative results of about 20,000 human single islet cells demonstrate the successful application of the protocol.

Here, we present a protocol to generate high-quality, large-scale transcriptome data of single cells from isolated human pancreatic islets using a droplet-based microfluidic single-cell RNA sequencing technology.

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to ...