This study was partially funded by Roche S.p.A. and the Italian Medicines Agency (AIFA).

NOTE: Perform all of the following steps under a sterilized fume hood according to Good Laboratory Practice (GLP).
1. Plasma Collection and Storage
<ol>
	<li>Collect 3 mL of peripheral blood sample in a K3E EDTA tube.</li>
	<li>Centrifuge the tube at room temperature at 1,880 x g for 15 min.</li>
	<li>Recover the supernatant plasma in aliquots of 450 &#181;L.</li>
	<li>Store the samples at -80 &#176;C until use. 
	NOTE: Process the peripheral blood tube within 2 h of collection. When recovering the plasma from the tube, do not disturb the lymphocyte layer.</li>
</ol>
2. Extraction of Circulating miRNAs from Plasma Samples (Table of Materials)
<ol>
	<li>Prepare all the required solutions and thaw the samples for the extraction steps.
	<ol>
		<li>Add 375 &#181;L of 2-mercaptoethanol to the 2x denaturating solution and mix well.</li>
		<li>Add 21 mL of ACS grade 100% ethanol to the miRNA wash solution I and mix well.</li>
		<li>Add 40 mL of ACS grade 100% ethanol to the wash solution 2/3 and mix well.</li>
		<li>Allow a plasma aliquot to thaw at room temperature and then begin the extraction steps.</li>
	</ol>
	</li>
	<li>Organic extraction
	<ol>
		<li>Transfer 400 &#181;L of the plasma sample into a RNase-free 2 mL tube.</li>
		<li>Add 400 &#181;L of 2x denaturating solution, mix by vortexing and briefly centrifuge.</li>
		<li>Add 4 &#181;L of 1 nM spike-in, mix by vortexing and briefly centrifuge.</li>
		<li>Add 800 &#181;L of acid phenol-chloroform.</li>
		<li>Vortex for 60 s and centrifuge at maximum speed (&#8805;10,000 x g) for 15 min.</li>
		<li>Transfer the upper aqueous phase to a fresh tube. Do not disturb the interphase. Note the volume recovered and discard the tube containing the non-acqueous phase. 
		NOTE: The 2x denaturing solution is stored at 4 &#176;C and may solidify at this temperature. Before use, warm the solution to 37 &#176;C, agitating occasionally for 10-15 min. Be careful to withdraw the bottom phase containing the acid phenol-chloroform, not the acqueous buffer that lies on top of the mixture. Preheat the elution solution or nuclease-free water to 95 &#176;C. Be careful to heat an adequate volume of solution (100 &#181;L per sample + 10% excess).</li>
	</ol>
	</li>
	<li>Small RNA enrichment
	<ol>
		<li>Add 1/3 volume of ethanol 100% to the aqueous phase from step 2.2.6 and mix thoroughly.</li>
		<li>Place a filter cartridge into a fresh collection tube for each sample.</li>
		<li>Transfer up to 700 &#181;L of lysate from step 2.3.1 and ethanol into the filter cartridge and centrifuge at 10,000 x g for 30 s.</li>
		<li>If there is &#62;700 &#181;L of mixture left, place the filtrate in a fresh tube and repeat step 2.3.3; repeat until all the mixture has passed through the filter cartridge.</li>
		<li>Measure the total volume of the flow-through.</li>
		<li>Add 2/3 volume of ethanol 100% to filtrate and mix thoroughly.</li>
		<li>Place a second filter cartridge in a fresh collection tube.</li>
		<li>Transfer up to 700 &#181;L of sample volume into the cartridge and centrifuge at 10,000 x g for 30 s. Discard the flow-through.</li>
		<li>Repeat step 2.3.8 until all the mixture has passed through the filter cartridge.</li>
		<li>Apply 700 &#181;L of miRNA wash solution 1 to the filter cartridge and centrifuge the filter cartridge with the collection tube at 10,000 x g for 15 s. Discard the flow-through. Place the filter cartridge in the same collection tube.</li>
		<li>Apply 500 &#181;L of wash solution 2/3 to the filter cartridge and centrifuge the filter cartridge with the collection tube at 10,000 x g for 15 s. Discard the flow-through. Place the filter cartridge in a fresh collection tube.</li>
		<li>Place the filter cartridge in a fresh collection tube and repeat step 2.3.11 with 500 &#181;L of wash solution 2/3. Discard the flow-through and transfer the filter cartridge to a fresh collection tube.</li>
		<li>Centrifuge the tubes with the filter cartridges at 10,000 x g for 1 min.</li>
		<li>Transfer the filter cartridge into a fresh 1.5 mL collection tube and add 100 &#181;L of preheated (95 &#176;C) elution solution or nuclease-free water.</li>
		<li>Centrifuge at 10,000 x g for 30 s to recover miRNAs and store at -80 &#176;C. 
		NOTE: A white precipitate may form in the wash solution 2/3. This is excess EDTA released from the solution. Avoid drawing up these crystals during the pipetting steps.</li>
	</ol>
	</li>
</ol>
3. Perform Reverse Transcription and miRNA Pre-amplification (Table of Materials)
<ol>
	<li>Perform polyadenylation reaction
	<ol>
		<li>Thaw all reagents on ice, then briefly vortex and centrifuge to spin down the contents. In addition, thaw 50% PEG 8000, allowing it to reach room temperature.</li>
		<li>Prepare the reaction mix in a 0.5 mL centrifuge tube, obtaining a final volume of 3 &#181;L of reaction cocktail for each sample. Use the following volumes, plus 10% excess volume for each reagent.</li>
		<li>Add 1.7 &#181;L of RNase-free water, 0.5 &#181;L of 10x Poly(A) buffer, 0.5 &#181;L of 10 mM ATP, and finally 0.3 &#181;L 5 U/&#181;L PolyA enzyme.</li>
		<li>Briefly vortex and centrifuge the cocktail mix to spin down the contents.</li>
		<li>Transfer 2 &#181;L of the sample to each well of the reaction plate or reaction tube and add 3 &#181;L of the reaction cocktail. Briefly vortex and centrifuge to spin down the contents.</li>
		<li>Place the plate/tubes into a thermal cycler and perform the following steps.</li>
		<li>Incubate at 37 &#176;C for 45 min (polyadenylation step).</li>
		<li>Incubate at 65 &#176;C for 10 min (stop reaction), with a following hold at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Perform the ligation reaction.
	<ol>
		<li>Prepare the reaction mix in a centrifuge tube with the following volumes for each sample and 10% of volume in excess.</li>
		<li>Add 0.4 &#181;L of RNase-free water, 3 &#181;L of 5x DNA ligase buffer, 0.6 &#181;L of 25x ligation adaptor, 4.5 &#181;L of 50% PEG 8000, and finally 1.5 &#181;L of RNA ligase.</li>
		<li>Briefly vortex and centrifuge the cocktail mix to spin down the content.</li>
		<li>Add 10 &#181;L of the mix to each well/reaction tube containing the poly(A) tailing product. Mix by a plate shaker at 1,900 rpm for 1 min or by briefly vortexing and spin down the contents.</li>
		<li>Place the plate/reaction tubes in a thermal cycler with the following settings: incubation at 60 &#176;C for 60 min, with a following hold at 4 &#176;C. 
		NOTE: 50% PEG 8000 is highly viscous. Use at room temperature, aspirating and dispensing slowly. After each aspiration and dispensing step, hold the plugger for 10 s to allow the reagent to flow up and down.</li>
	</ol>
	</li>
	<li>Perform the reverse transcription reaction.
	<ol>
		<li>Prepare the reaction mix in a centrifuge tube, with the following volumes for each sample and 10% of volume in excess.</li>
		<li>Add 3.3 &#181;L of RNase-free water, 6 &#181;L of 5x RT buffer, 1.2 &#181;L of dNTP mix (25 mM each), 1.5 &#181;L of 20x Universal RT primers, and finally 3 &#181;L of 10x RT enzyme mix.</li>
		<li>Briefly vortex and centrifuge the cocktail mix to spin down the contents.</li>
		<li>Add 15 &#181;L of the mix to each well/reaction tube containing the ligation product. Mix by a plate shaker at 1,900 rpm for 1 min or by briefly vortexing and spin down the contents.</li>
		<li>Place the plate/reaction tubes in a thermal cycler with the following settings.</li>
		<li>Incubate at 42 &#176;C for 15 min (reverse transcription).</li>
		<li>Incubate at 85 &#176;C for 5 min (stop reaction), with a following hold at 4 &#176;C. 
		NOTE: The RT product can now be stored at -20 &#176;C and will remain stable for up to 2 months.</li>
	</ol>
	</li>
	<li>Perform the miR-Amp reaction (Table of Materials).
	<ol>
		<li>Prepare the reaction mix in a centrifuge tube with the following volumes for each sample and 10% of volume in excess.</li>
		<li>Add 17.5 &#181;L of RNase-free water, 25 &#181;L of the 2x miR-Amp master mix, and 2.5 &#181;L of 20x miR-Amp primer mix.</li>
		<li>Briefly vortex and centrifuge the cocktail mix to spin down the contents.</li>
		<li>For each sample, transfer 45 &#181;L of the reaction mix to each well of a new reaction plate or a reaction tube. Add 5 &#181;L of the RT product to each well or reaction tube. Mix by a plate shaker at 1,900 rpm for 1 min or by briefly vortexing and spin down the contents.</li>
		<li>Place the plate/reaction tubes in a thermal cycler and run as shown in Table 1.</li>
	</ol>
	</li>
</ol>
4. Perform Real-time PCR
<ol>
	<li>Dilute each cDNA sample 1:10 in 0.1x TE buffer.</li>
	<li>Calculate the number of wells/replicates for each sample. Prepare the reaction mix in a centrifuge tube with the following volumes for each sample, adding 10% of volume in excess.</li>
	<li>Add 4 &#181;L of RNase-free water, 10 &#181;L of 2x fast advanced master mix (Table of Materials), and finally 5 &#181;L of diluted cDNA.</li>
	<li>Mix by vortexing and briefly centrifuge to spin down the contents.</li>
	<li>Transfer 19 &#181;L of reaction mix to each well, seal the plate and briefly centrifuge.</li>
	<li>Perform the thermal protocol (Table 2) on a RT-PCR system.</li>
</ol>

<ol>
	<li>Luo, H. -. Y., Xu, R. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predictive+and+prognostic+biomarkers+with+therapeutic+targets+in+advanced+colorectal+cancer.">Predictive and prognostic biomarkers with therapeutic targets in advanced colorectal cancer.</a> World journal of gastroenterology. 20 (14), 3858-3874 (2014).</li><li>Toiyama, Y., Okugawa, Y., Fleshman, J., Richard, C., Goel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+as+potential+liquid+biopsy+biomarkers+in+colorectal+Cancer:+A+systematic+review.">MicroRNAs as potential liquid biopsy biomarkers in colorectal Cancer: A systematic review.</a> BBA Reviews on Cancer. 5 (6), (2018).</li><li>Kok, M. G. M., Halliani, A., Moerland, P. D., Meijers, J. C. M., Creemers, E. E., Pinto-Sietsma, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normalization+panels+for+the+reliable+quantification+of+circulating+microRNAs+by+RT-qPCR.">Normalization panels for the reliable quantification of circulating microRNAs by RT-qPCR.</a> FASEB Journal. 29 (9), 3853-3862 (2015).</li><li>Marabita, F., De Candia, P., Torri, A., Tegn&#233;r, J., Abrignani, S., Rossi, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normalization+of+circulating+microRNA+expression+data+obtained+by+quantitative+real-time+RT-PCR.">Normalization of circulating microRNA expression data obtained by quantitative real-time RT-PCR.</a> Briefings in Bioinformatics. 17 (2), 204-212 (2016).</li><li>Danese, E., 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=Reference+miRNAs+for+colorectal+cancer:+Analysis+and+verification+of+current+data.">Reference miRNAs for colorectal cancer: Analysis and verification of current data.</a> Scientific Reports. 7 (1), 1-12 (2017).</li><li>Zheng, 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=Identification+and+validation+of+reference+genes+for+qPCR+detection+of+serum+microRNAs+in+colorectal+adenocarcinoma+patients.">Identification and validation of reference genes for qPCR detection of serum microRNAs in colorectal adenocarcinoma patients.</a> PLoS ONE. 8 (12), 1-10 (2013).</li><li>Lv, 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=Optimization+of+the+Original+TRIzol-Based+Technique+Improves+the+Extraction+of+Circulating+MicroRNA+from+Serum+Samples.">Optimization of the Original TRIzol-Based Technique Improves the Extraction of Circulating MicroRNA from Serum Samples.</a> Clinical laboratory. 61 (12), 1953-1960 (2015).</li><li>Tan, G. W., Khoo, A. S. B., Tan, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+extraction+kits+and+RT-qPCR+systems+adapted+to+high-throughput+platform+for+circulating+miRNAs.">Evaluation of extraction kits and RT-qPCR systems adapted to high-throughput platform for circulating miRNAs.</a> Scientific reports. 5, 9430 (2015).</li><li>Altman, D. G., McShane, L. M., Sauerbrei, W., Taube, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reporting+Recommendations+for+Tumor+Marker+Prognostic+Studies+(REMARK):+explanation+and+elaboration.">Reporting Recommendations for Tumor Marker Prognostic Studies (REMARK): explanation and elaboration.</a> PLoS medicine. 9 (5), (2012).</li><li>Tiberio, P., Callari, M., Angeloni, V., Daidone, M. G., Appierto, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+in+using+circulating+miRNAs+as+cancer+biomarkers.">Challenges in using circulating miRNAs as cancer biomarkers.</a> BioMed Research International. 2015, (2015).</li><li>Kroh, E. M., Parkin, R. K., Mitchell, P. S., Tewari, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+circulating+microRNA+biomarkers+in+plasma+and+serum+using+quantitative+reverse+transcription-PCR+(qRT-PCR).">Analysis of circulating microRNA biomarkers in plasma and serum using quantitative reverse transcription-PCR (qRT-PCR).</a> Methods (San Diego, Calif). 50 (4), 298-301 (2010).</li><li>Cheng, H. H., 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=Plasma+Processing+Conditions+Substantially+Influence+Circulating+microRNA+Biomarker+Levels.">Plasma Processing Conditions Substantially Influence Circulating microRNA Biomarker Levels.</a> PLoS ONE. 8 (6), 1-11 (2013).</li><li>Moret, I., 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=Assessing+an+improved+protocol+for+plasma+microRNA+extraction.">Assessing an improved protocol for plasma microRNA extraction.</a> PloS one. 8 (12), (2013).</li><li>Khoury, S., Ajuyah, P., Tran, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+small+noncoding+RNAs+from+human+serum).">Isolation of small noncoding RNAs from human serum).</a> Journal of visualized experiments JoVE. (88), e51443 (2014).</li><li>Schwarzenbach, H., Nishida, N., Calin, G. A., Pantel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+relevance+of+circulating+cell-free+microRNAs+in+cancer.">Clinical relevance of circulating cell-free microRNAs in cancer.</a> Nature Reviews Clinical Oncology. 11 (3), 145-156 (2014).</li></ol>

The authors have no conflicts of interest to disclose.

miRNAs are small non-coding RNAs (18-25 nucleotides in length) capable of binding the 3&#39; UTR region of their target messenger RNA and inhibiting and/or degrading it. They can thus be considered gene expression regulators at the translational level. In the last decade, several miRNAs have been correlated with cancer initiation and development, making them useful clinical biomarkers for diagnosis, prognosis and therapy. Protected by RNases, miRNAs are present in a stable form in biological fluids such as blood, plasma, serum, urine and saliva, which has led to a growing interest in their potential usefulness in clinical practice10.
Despite this, the evaluation of circulating miRNAs is a challenging task and difficulties regarding sample collection, target extraction, platform choice and global normalization are hotly debated topics within the scientific community.
For sample collection and storage, pre-analytical variables are closely linked to plasma handling and processing. We focused on plasma rather than serum samples given that there is ample evidence of higher miRNA concentrations in the latter, possibly due to the release of these molecules during the coagulation process10,11,12. To address the issue of the release of cellular nucleic acids deriving from blood cell lysis, we centrifuged the samples within 2 h of blood collection. We also used K3E EDTA tubes because other anti-coagulants are known to interfere with amplification steps (i.e., heparin) and may trigger hemolysis (i.e., citrate). As it is critical to avoid platelet and lymphocyte contamination in the pre-analytical phase, we removed the plasma from the tube very slowly and did not aspirate the last ~50 &#181;L of the sample from the tube.
Various studies have shown the superiority of column-based extraction methods over guanidine/phenol/chloroform-based protocols11,13, but Khoury et al. effectively isolated good quality small RNAs from serum using such reagents without contamination14. The commercial kit we used sequentially performs both extraction methods, permitting suitable miRNA yields in terms of spike-in concentrations. We only performed one modification with respect to the manufacturer&#39;s instructions, i.e., fresh collection tubes were always used after each filter cartridge washing step to eliminate/reduce the risk of reagent contamination. In order to identify the best spike-in concentration in our samples and thus avoid interference with subsequent reactions, we first performed the entire protocol without adding the cel-miR-39 during the extraction step to evaluate the median expression levels of our targets. We then performed de novo miRNA extraction from plasma samples of the same patient using a serial dilution of cel-miR-39 to identify the best concentration to add to the plasma samples of our case series (Figure 4). It must, however, be underlined that the optimal cel-miR-39 concentration, identified for only one sample, may not necessarily be the best concentration for the overall case series because of a series of internal variables such as age, gender or life habits, which could influence absolute miRNA expression within blood.
miRNA reverse transcription and amplification represent 2 critical steps. Of the two main techniques for miRNA detection (i.e., qRT-PCR and microarray), we chose qRT-PCR given its high sensitivity and our need to evaluate only a selected panel of miRNAs (a major limitation of this methodology is its low throughput)15. We used reverse transcription chemistry based on 3&#39; polyA tailing and 5&#39; ligation adaptor sequences to extend the mature miRNA and permit annealing of universal RT primers. Based on single base-pair recognition, the 5&#39; ligation adaptor could help to avoid common biases such as those linked to 5&#39; mismatch. This kit also includes a pre-amplification step that is required for low yield samples, such as plasma. Although necessary, this step could introduce some amplification biases before the detection step.
In this protocol, we analyzed a panel of 24 selected miRNAs correlated with angiogenesis. In particular, we chose pre-spotted probe custom plates and followed the manufacturer&#39;s instructions for the qRT-PCR runs. The main issues relating to miRNA qRT-PCR are the choice of reference genes and their subsequent data analysis normalization.
Although numerous studies have been conducted to identify the best miRNA to use for normalization, no uniform consensus has been reached. To address this limitation, we carried out a literature search to identify the circulating miRNAs that can be used as reference genes in our case series.
We also selected the 4 miRNAs with strong evidence of stable expression in plasma samples from mCRC patients and tested them in a smaller part of our case series. The 2 most stable miRNAs were identified by GeNorm analysis as reference housekeeping genes.
In conclusion, the evaluation of circulating miRNAs in peripheral blood samples has a number of known difficulties. However, we believe that the methodologies we used are reliable and robust, allowing for an in-depth and accurate study of these biomarkers which have the potential to change the treatment scenario for colorectal cancer.

CRC represents the third most frequently diagnosed malignancy and the fourth cause of cancer-related death worldwide. To date, bevacizumab (B), a monoclonal antibody directed against vascular-endothelial growth factor (VEGF), and cetuximab (C) or panitumumab (P), monoclonal antibodies directed against epidermal growth factor receptor (EGFR), have been approved for first-line treatment in combination with chemotherapy (CT) regimens.
Mutations in K-RAS and N-RAS genes are the only clinically useful biomarkers capable of identifying patients who are least likely to benefit from anti-EGFR-based CT. Although several studies have been conducted to search for biomarkers that are predictive of response to B-based CT, there is still a substantial lack of reliable and effective drugs for use in clinical practice1.
miRNAs are small RNAs (18-25 nucleotides in length) that regulate the translation of target genes and play a crucial role in numerous physiopathological processes, including embryogenesis and carcinogenesis. These molecules are highly stable in biological fluids such as plasma/serum, urine and sputum, which renders them robust biomarkers to use for non-invasive sampling2. Using a panel of miRNAs involved in the angiogenic pathway, we aimed to identify new circulating biomarkers capable of predicting clinical outcome in patients with metastatic CRC (mCRC) treated with a B-based CT regimen.
We analyzed a series of 52 mCRC patients treated with B-based CT within the prospective multicenter randomized phase III trial &#34;Italian Trial in Advanced Colorectal Cancer&#34; (ITACa). The present protocol was approved by the Local Ethics Committee (Comitato Etico Area Vasta e Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, no. 674) on 19th September 2007. All patients gave informed consent before blood sample collection. For each patient, venous blood samples were collected before treatment and at the first clinical evaluation (after 8 weeks) to evaluate baseline miRNA expression and its modulation during treatment in relation to patient outcome.
Through a search of the literature, we selected 21 miRNAs correlated with the angiogenic process that are known to be detectable in human plasma: hsa-miR-107, hsa-miR-126-3p, hsa-miR-145-5p, hsa-miR-194-5p, hsa-miR-199a-5p, hsa-miR-200b-3p, hsa-miR-20b-5p, hsa-miR-21-5p, hsa-miR-210-3p, hsa-miR-221-3p, hsa-miR-24-3p, hsa-miR-27a-3p, hsa-miR-29b-3p, hsa-miR-335-5p, hsa-miR-424-5p, hsa-miR-497-5p, hsa-miR-520d-3p, hsa-miR-92a-3p, hsa-miR-17-5p and hsa-miR-155-5p. We also selected hsa-miR-223-3p and hsa-mir-484 for endogenous normalization3,4,5,6, and cel-miR-39 purified from C. elegans as a spike-in for exogenous normalization. All data normalizations were performed using the 2 &#770; &#772;(&#916;&#916;Ct) method.
The detection of circulating miRNAs presents some technical difficulties because the molecules are present at very low levels in plasma and their amplification is chemically challenging given their short sequence. For these reasons, we selected a procedure that considers both organic extraction with phenol and a glass-fiber column-based methodology. Circulating miRNA extraction is a hot topic in the field of liquid biopsy, and we selected a commercial kit that has shown to be one of the most reliable in terms of amount and quality of recovered yields7,8. We selected a protocol to reverse transcribe miRNAs by the addition of an adapter at 5&#39; and a poly(A) tail to 3&#39; of the mature miRNA to enhance the selectivity and specificity of the reaction.
Given the robustness of the method, we designed custom plates with pre-spotted probes for RT-PCR to assess each sample in duplicate and analyzed 2 patients within each plate, as shown in Figure 1.

<table><tbody><tr><td>Rnase-free Safe-lock 1.5 mL tubes</td><td>Eppendorf</td><td>0030 123.328</td><td></td></tr><tr><td>Rnase-free Safe-lock 2 mL tubes</td><td>Eppendorf</td><td>0030 123.344</td><td></td></tr><tr><td>Rnase-free 20 &amp;micro;L tips</td><td>Starlab</td><td>S1123-1810</td><td></td></tr><tr><td>Rnase-free 200 &amp;micro;L tips</td><td>Starlab</td><td>S1120-8810</td><td></td></tr><tr><td>Rnase-free 1,000 &amp;micro;L tips</td><td>Starlab</td><td>S1122-1830</td><td></td></tr><tr><td>mirVana PARIS RNA and Native Protein Purification Kit</td><td>Thermo Fisher</td><td>AM1556</td><td></td></tr><tr><td>TaqMan Advanced miRNA cDNA Synthesis Kit</td><td>Thermo Fisher</td><td>A28007</td><td></td></tr><tr><td>100% ethanol anidrous ACS grade</td><td>Carlo Erba Reagents</td><td>414605</td><td></td></tr><tr><td>2-mercapto-ethanol</td><td>Sigma-Aldrich</td><td>M3148</td><td></td></tr><tr><td>TaqMan Fast Advanced Master Mix</td><td>Thermo Fisher</td><td>4444558</td><td></td></tr><tr><td>TaqMan Advanced miRNA Assays</td><td>Thermo Fisher</td><td>A25576</td><td></td></tr><tr><td>7500 Real-Time PCR System</td><td>Applied Biosystems</td><td>4406984</td><td></td></tr><tr><td>0.2-2, 1-10, 2-20, 20-200, 100-1,000 &amp;micro;L laboratory pipettes</td><td></td><td></td><td></td></tr><tr><td>Benchtop microcentrifuge</td><td></td><td></td><td></td></tr><tr><td>Vortex</td><td></td><td></td><td></td></tr><tr><td>Benchtop heating block</td><td></td><td></td><td></td></tr><tr><td>Fume hood</td><td></td><td></td><td></td></tr><tr><td>0.2 mL PCR tubes</td><td></td><td></td><td></td></tr></tbody></table>

detection of a circulating microrna custom panel in patients with metastatic colorectal cancer

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful biomarkers for clinical practice. Here, we present a protocol to extract, reverse transcribe and evaluate the expression levels of circulating miRNAs from plasma samples of patients with colorectal cancer (CRC). microRNAs (miRNAs) are a class of non-coding RNAs of 18-25 nucleotides in length that regulate the expression of target genes at the translational level and play an important role, including that of pro- and anti-angiogenic function, in the physiopathology of various organs. miRNAs are stable in biological fluids such as serum and plasma, which renders them ideal circulating biomarkers for cancer diagnosis, prognosis and treatment decision-making and monitoring. Circulating miRNA extraction was performed using a rapid and effective method that involves both organic and column-based methodologies. For miRNA retrotranscription, we used a multi-step procedure that considers polyadenylation at 3&#39; and the ligation of an adapter at 5&#39; of the mature miRNAs, followed by random miRNA pre-amplification. We selected a 24-miRNA custom panel to be tested by quantitative real-time polymerase chain reaction (qRT-PCR) and spotted the miRNA probes on array custom plates. We performed qRT-PCR plate runs on a real-time PCR System. Housekeeping miRNAs for normalization were selected using GeNorm software (v. 3.2). Data were analyzed using Expression suite software (v 1.1) and statistical analyses were performed. The method proved to be reliable and technically robust and could be useful to evaluate biomarker levels in liquid samples such as plasma and/or serum.

We analyzed a panel of angiogenesis-related circulating miRNAs in relation to progression-free survival (PFS), overall survival (OS) and objective response rate (ORR) in a 52 mCRC patients treated with B-based CT. The evaluation of such biomarkers in plasma samples is challenging because of technical difficulties. We used established methods for miRNA extraction from patient plasma samples, reverse transcription and pre-amplification. Following the manufacturer&#39;s instructions and with only a few modifications required, especially in the extraction steps, we successfully evaluated all our targets in the patient population (Figure 2).
In particular, we analyzed 2 plasma samples/patient, correlating baseline and therapy-modified miRNA expression with patient outcome.
We performed serial dilutions of cel-miR-39 at 0.05 nM, 0.5 nM, 5nM and 5 pM in 800 &#181;L of plasma sample (400 &#181;L of plasma plus 400 &#181;L of 2x denaturating solution) to determine the spike-in concentration to use. As shown in Figure 4, the exogenous control showed threshold cycle (Ct) values that were congruent with the serial dilutions, indicating the robustness of the entire protocol (i.e., extraction, reverse transcription and qRT-PCR evaluation). Moreover, these serial dilutions enabled us to select the spike-in concentration that would not interfere with the reverse-transcription of all target miRNAs.
This method allowed us to correlate baseline miRNAs with clinical pathological features. In particular, we found that hsa-miR-199a-5p, hsa-miR-335-5p and hsa-miR-520d-3p were significantly upregulated in left-sided with respect to right-sided lesions (p&#160;= 0.03, p&#160;= 0.006 and p&#160;= 0.008, respectively). Conversely, hsa-miR-21-5p was significantly downregulated in RAS-mutated patients (K-RAS, p&#160;= 0.01 and N-RAS, p&#160;= 0.008), whereas hsa-miR-221-3p was upregulated (p&#160;= 0.05, p&#160;= 0.01, K- and N-RAS, respectively).
Comparing the expression levels of miRNA between baseline and first clinical evaluation, we observed that an increase in hsa-miR-155-5p expression levels was associated with shorter PFS (p&#160;= 0.04) and OS (p&#160;= 0.02). In particular, in patients with a &#8805;30% increase in hsa-miR-155-5p, PFS was 9.5 months (95% CI, 6.8-18.7) and OS was 15.9 months (95% CI, 8.4-not reached) compared to 22.3 months (95% CI, 10.2-25.5) and 42.9 months (95% CI, 24.8-not reached) for those with a &#60;30% increase (Figure 3). The median value of variation in the case series (30%) was set as the cutoff point. Circulating basal levels of miRNAs were dichotomized into &#34;high&#34; or &#34;low&#34; according to median values9.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58615/58615fig1.jpg" /> 
Figure 1: Design of custom plate layout. Probes were pre-spotted in each well. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58615/58615fig1large.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/58615/58615fig2.jpg" /> 
Figure 2: Examples of qRT-PCR amplification plots. (A) Amplification plot of a single qRT-PCR custom plate. All markers were evaluable in both single plate samples. (B) qRT-PCR of hsa-miR-17-5p in the overall case series. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58615/58615fig2large.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/58615/58615fig3.jpg" /> 
Figure 3: Experimental and clinical results relative to hsa-miR-155-5p. (A) Ct values for hsa-miR-155-5p. Amplification plot of hsa-miR-155-5p in the overall case series. (B) PFS and OS of patients with a &#8805;30% or &#60;30% increase in circulating hsa-miR-155-5p. PFS was calculated as the time from the date of randomization to the date of the first documented evidence of tumor progression, last tumor assessment or death in the absence of disease progression. OS was calculated as the time from the date of randomization to the date of death from any cause or last follow-up. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58615/58615fig3large.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/58615/58615fig4.jpg" /> 
Figure 4: Experimental results forcel-miR-39 serial dilution. (A) qRT-PCR for serial dilutions of cel-miR-39, with (B) relative Cts. Dilutions were performed at 5 nM, 0.5 nM, 0.05 nM and 5 pM in plasma samples from the same patient. Such assay linearity enabled us to select the best concentration to use in the overall case series. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58615/58615fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Step</td>
			<td>Temperature</td>
			<td>Duration</td>
			<td>Cycles</td>
		</tr>
		<tr>
			<td>Enzyme activation</td>
			<td>95 &#176;C</td>
			<td>5 min</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Denature</td>
			<td>95 &#176;C</td>
			<td>3 s</td>
			<td rowspan="2">14</td>
		</tr>
		<tr>
			<td>Anneal/extend</td>
			<td>60 &#176;C</td>
			<td>30 s</td>
		</tr>
		<tr>
			<td>Stop reaction</td>
			<td>99 &#176;C</td>
			<td>10 min</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Hold</td>
			<td>4 &#176;C</td>
			<td>Hold</td>
			<td>&#8734;</td>
		</tr>
	</tbody>
</table>
Table 1: Thermal protocol of mir-AMP reaction.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Step</td>
			<td>Temperature</td>
			<td>Duration</td>
			<td>Cycles</td>
		</tr>
		<tr>
			<td>Enzyme activation</td>
			<td>95 &#176;C</td>
			<td>20 s</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Denature</td>
			<td>95 &#176;C</td>
			<td>3 s</td>
			<td rowspan="2">40</td>
		</tr>
		<tr>
			<td>Anneal/extend</td>
			<td>60 &#176;C</td>
			<td>30 s</td>
		</tr>
		<tr>
			<td>Hold</td>
			<td>4 &#176;C</td>
			<td>Hold</td>
			<td>&#8734;</td>
		</tr>
	</tbody>
</table>
Table 2: Thermal protocol of qRT-PCR.

Watch this Scientific Journal Video about Detection of a Circulating MicroRNA Custom Panel in Patients with Metastatic Colorectal Cancer at JoVE.com

Detection of a Circulating MicroRNA Custom Panel in Patients with Metastatic Colorectal Cancer

We present a protocol to evaluate the expression levels of circulating microRNA (miRNAs) in plasma samples from cancer patients. In particular, we used a commercially available kit for circulating miRNA extraction and reverse transcription. Finally, we analyzed a panel of 24 selected miRNAs using real-time pre-spotted probe custom plates.

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful ...

detection-circulating-microrna-custom-panel-patients-with-metastatic

Nanyang Technological University

Research

JoVE Journal

Cancer Research

5.4K Views.  Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS. Deze methode kan helpen bij het beantwoorden van belangrijke vragen op het gebied van translationeel onderzoek, zoals de relatie tussen circulerende biomarkers en de patiëntenprognose. Het belangrijkste voordeel van deze techniek is de kans om de expressie van een paneel van circulerende biomarkers te evalueren met een eenvoudige en betrouwbare methodologie met een lage hoeveelheid inputmateriaal. De implicaties van deze techniek uit te breiden tot therapie van kankerpatiënten, omdat het ko...

Video: Detection of a Circulating MicroRNA Custom Panel in Patients with Metastatic Colorectal Cancer

Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in a single reaction. The miRNA assay counts transcripts by directly imaging and digitally counting miRNA molecules that are labeled with color-coded fluorescent barcoded probe sets (a reporter probe and a capture probe). Barcodes are hybridized directly to mature miRNAs that have been elongated by ligating a unique oligonucleotide tag (miRtag) to the 3&#39; end. Reverse transcription and amplification of the transcripts are not required. Reporter probes contain a sequence of six color positions populated using a combination of four fluorescent colors. The four colors over six positions are used to construct a gene-specific color barcode sequence. Post-hybridization processing is automated on a robotic prep station. After hybridization, the excess probes are washed away, and the tripartite structures (capture probe-miRNA-reporter probe) are fixed to a streptavidin-coated slide via the biotin-labeled capture probe. Imaging and barcode counting is done using a digital analyzer. The immobilized barcoded miRNAs are visualized and imaged using a microscope and camera, and the unique barcodes are decoded and counted. Data quality control (QC), normalization, and analysis are facilitated by a custom-designed data processing and analysis software that accompanies the assay software. The assay demonstrates high linearity over a broad range of expression, as well as high sensitivity. Sample and assay preparation does not involve enzymatic reactions, reverse transcription, or amplification; has few steps; and is largely automated, reducing investigator effects and resulting in high consistency and technical reproducibility. Here, we describe the application of this technology to identifying circulating miRNA perturbations in irritable bowel syndrome.

We describe the use of a multiplexed high-throughput gene expression platform that quantitates gene expression by barcoding and counting molecules in biological substrates without the need for amplification. We used the platform to quantitate microRNA (miRNA) expression in whole blood in subjects with and without irritable bowel syndrome.

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in ...

Genetics

MicroRNA Based Liquid Biopsy: The Experience of the Plasma miRNA Signature Classifier (MSC) for Lung Cancer Screening

The development of a minimally invasive test, such as liquid biopsy, for early lung cancer detection in its preclinical phase is crucial to improve the outcome of this deadly disease. MicroRNAs (miRNAs) are tissue specific, small, non-coding RNAs regulating gene expression, which may act as extracellular messengers of biological signals derived from the cross-talk between the tumor and its surrounding microenvironment. They could thus represent ideal candidates for early detection of lung cancer. In this work, a methodological workflow for the prospective validation of a circulating miRNA test using custom made microfluidic cards and quantitative Real-Time PCR in plasma samples of volunteers enrolled in a lung cancer screening trial is proposed. In addition, since the release of hemolysis-related miRNAs and more general technical issues may affect the analysis, the quality control steps included in the standard operating procedures are also presented. The protocol is reproducible and gives reliable quantitative results; however, when using large clinical series, both pre-analytical and analytical features should be cautiously evaluated.

MicroRNA Based Liquid Biopsy: The Experience of the Plasma miRNA Signature Classifier MSC for Lung Cancer Screening

Here, we present the detailed protocol adopted in the BioMILD screening trial to perform the circulating microRNA signature classifier test for early lung cancer detection.

The development of a minimally invasive test, such as liquid biopsy, for early lung cancer detection in its preclinical phase is crucial to improve the ...

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal gastric mucosa. Recently, we detected CDH1a transcript variant in fresh-frozen tumor tissues obtained from patients with GC. The expression of this variant in tissue samples was investigated by the chip-based digital PCR (dPCR) approach presented here. dPCR offers the potential for an accurate, robust, and highly sensitive measurement of nucleic acids and is increasingly utilized for many applications in different fields. dPCR is capable of detecting rare targets; in addition, dPCR offers the possibility for absolute and precise quantification of nucleic acids without the need for calibrators and standard curves. In fact, the reaction partitioning enriches the target from the background, which improves amplification efficiency and tolerance to inhibitors. Such characteristics make dPCR an optimal tool for the detection of the CDH1a rare transcript.

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

The manuscript describes a chip-based digital PCR assay to detect a rare CDH1 transcript variant (CDH1a) in fresh-frozen normal and tumor tissues obtained from patients with gastric cancer.

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal ...

High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this clinical challenge, tumor xenograft mouse models using long-established human carcinoma cell lines and many transgenic mouse models with tumors have been developed as preclinical models. They partially mimic the features of human carcinomas, but often fail to recapitulate the key aspects of human malignancies including invasion and metastasis. Thus, alternative models that better represent the malignant progression in human CRC have long been awaited.
We herein show generation of patient-derived tumor xenografts (PDXs) by subcutaneous implantation of small CRC fragments surgically dissected from a patient. The colon PDXs develop and histopathologically resemble the CRC in the patient. However, few spontaneous micrometastases are detectable in conventional cross-sections of affected distant organs in the PDX model. To facilitate the detection of metastatic dissemination into distant organs, we extracted the tumor organoid cells from the colon PDXs in culture and infected them with GFP lentivirus prior to injection into highly immunodeficient NOD/Shi-scid IL2R&#947;null (NOG) mice. Orthotopically injected PDX-derived CRC organoid cells consistently form primary tumors positive for GFP in recipient mice. Moreover, spontaneously developing micrometastatic colonies expressing GFP are notably detected in the lungs of these mice by fluorescence microscopy. Moreover, intrasplenic injection of CRC organoids frequently produces hepatic colonization. Taken together, these findings indicate GFP-labelled PDX-derived CRC organoid cells to be visually detectable during a multistep process termed the invasion-metastasis cascade. The described protocols include the establishment of PDXs of human CRC and 3D culture of the corresponding CRC organoid cells transduced by GFP lentiviral particles.

To allow highly sensitive detection of the disseminating human colorectal cancer (CRC) cells colonizing tissues, we herein show a protocol for efficient transduction of green fluorescent protein (GFP) lentiviral particles into PDX-derived CRC organoid cells prior to their injection into recipient mice, with stereo-fluorescence microscopic observation.

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this ...

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

Medicine

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been linked to a disease state and has the potential to be used as disease biomarker. A sensitive and accurate method for circulating microRNA quantification using a dye-based chemistry and droplet digital PCR technology has been recently developed. Specifically, using Locked Nucleic Acid (LNA)-based miRNA-specific primers with a green fluorescent DNA-binding dye in a compatible droplet digital PCR system it is possible to obtain the absolute quantification of specific miRNAs. Here, we describe how performing this technique to assess miRNA amount in biological fluids, such as plasma and serum, is both feasible and effective.

A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been ...

Bioengineering

Exosomal miRNA Analysis in Non-small Cell Lung Cancer (NSCLC) Patients' Plasma Through qPCR: A Feasible Liquid Biopsy Tool

The discovery of alterations in the EGFR and ALK genes, amongst others, in NSCLC has driven the development of targeted-drug therapy using selective tyrosine kinase inhibitors (TKIs). To optimize the use of these TKIs, the discovery of new biomarkers for early detection and disease progression is mandatory. These plasma-isolated exosomes can be used as a non-invasive and repeatable way for the detection and follow-up of these biomarkers. One ml of plasma from 12 NSCLC patients, with different mutations and treatments (and 6 healthy donors as controls), were used as exosome sources. After RNAse treatment, in order to degrade circulating miRNAs, the exosomes were isolated with a commercial kit and resuspended in specific buffers for further analysis. The exosomes were characterized by western blotting for ALIX and TSG101 and by transmission electron microscopy (TEM) analysis, the standard techniques to obtain biochemical and dimensional data of these nanovesicles. Total RNA extraction was performed with a high yield commercial kit. Due to the limited miRNA-content in exosomes, we decided to perform retro-transcription PCR using an individual assay for each selected miRNA. A panel of miRNAs (30b, 30c, 103, 122, 195, 203, 221, 222), all correlated with NSCLC disease, were analyzed taking advantage of the remarkable sensitivity and specificity of Real-Time PCR analysis; mir-1228-3p was used as endogenous control and data were processed according to the formula 2-&#916;&#916;ct 13. Control values were used as baseline and results are shown in logarithmic scale.

Exosomal miRNA Analysis in Non-small Cell Lung Cancer NSCLC Patients' Plasma Through qPCR: A Feasible Liquid Biopsy Tool

This protocol describes the feasibility to perform miRNA profiling in exosomes, released in plasma of NSCLC patients, through a commercial exosome isolation kit with Proteinase K and RNAse treatments, in order to avoid circulating miRNAs contamination and evaluate their biomarker features in NSCLC.

The discovery of alterations in the EGFR and ALK genes, amongst others, in NSCLC has driven the development of targeted-drug therapy using selective ...

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA (Locked Nucleic Acid) probe. The probe was recognized by anti-DIG alkaline phosphatase antibodies and later developed using alkaline phosphatase substrate producing fluorescence signals. Here we utilized miRNA in situ hybridization (MISH) technique to analyze expression of miR-489 in tissues from breast cancer patients. This technique can detect the localization of miRNA of interest in individual tissue samples. This technique can be used to compare the expression of desired miRNA in tumor tissue with that in adjacent normal tissue and to identify the specific structures responsible for expressing this miRNA. This technique can be very useful in answering certain clinical questions, such as role of specific miRNA in the development of cancer. Our results indicate that mammary epithelial cells express significantly higher levels of miR-489 than adjacent tumor cells.

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

Here we present a protocol to detect miRNA expression in breast cancer patient samples using miRNA in situ hybridization.

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA ...

Detection of a Circulating MicroRNA Custom Panel in Patients with Metastatic Colorectal Cancer

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