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 b...

<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,...

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 &#181;L tips</td>
			<td>Starlab</td>
			<td>S1123-1810</td>
			<td></td>
		</tr>
		<tr>
			<td>Rnase-free 200 &#181;L tips</td>
			<td>Starlab</td>
			<td>S1120-8810</td>
			<td></td>
		</tr>
		<tr>
			<td>Rnase-free 1000 &#181;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, 100-1000 &#181;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...

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 ...

This method can help answer key questions in the translational research field, such as the relationship between circulating biomarkers and the patient prognosis. The main advantage of this technique is the chance to evaluate the expression of a panel of circulating biomarkers with an easy and reliable methodology with low amount of input material. The implications of this technique extend toward therapy of cancer patients because it could identify circulating biomarkers useful in decision-making and treatment monitoring of patients.Also, this method can provide insight into circulating biomarkers analysis. It can also be applied to other systems, such as tissue biomarker expression analysis for molecular characterization of disease. Visual demonstration of this method is critical, as the extraction steps are difficult to learn.To begin, transfer 400 microliters of the plasma sample into an RNase-free, two-milliliter tube. Then, add 400 microliters of denaturing solution to the tube. Add four microliters of one-nanomolar spike-in.After mixing the solution, add 800 microliters of acid phenol-chloroform. Vortex the solution for 60 seconds, and centrifuge at maximum speed for 15 minutes. Next, transfer the lysate contained in the upper aqueous phase to a clean tube, taking care not to disturb the interphase.Measure the volume of the aqueous phase recovered, and add 1/3 volume of ethanol to the lysate. Then, place a filter cartridge into a fresh collection tube. Transfer up to 700 microliters of the ethanol-lysate mixture to the filter cartridge.Centrifuge the filter cartridge for 30 seconds at 10, 000 times gravity. Next, measure the total volume of the flow-through. Then, add 2/3 of the volume of ethanol to the filtrate, and mix well.Place a second filter cartridge into a fresh collection tube. Then, transfer up to 700 microliters of the sample to the cartridge, and centrifuge at 10, 000 times gravity for 30 seconds before discarding the flow-through. Add 700 microliters of microRNA wash solution to the filter cartridge, and centrifuge it with a collection tube at 10, 000 times gravity for 15 seconds.Discard the flow-through, and put the filter cartridge back into the same tube. After this, move the filter cartridge to a new collection tube. Then, add 100 microliters of preheated elution solution to the cartridge.Centrifuge the filter cartridge at 10, 000 times gravity for 30 seconds to recover the microRNAs. Prepare the polyadenylation reaction mix cocktail according to the text protocol. Transfer two microliters of the sample to each well of a reaction plate.Then, add three microliters of the reaction cocktail to each well. Vortex and centrifuge the plate briefly to spin down the contents. Next, place the plate in a thermal cycler, and follow the procedure outlined in the text protocol.Prepare the ligation reaction mix cocktail in a centrifuge tube. Next, add 10 microliters of the cocktail to each well of the reaction plate. Then, mix the contents of the reaction plate on a shaker at 1, 900 rpm for one minute, followed by a brief centrifuge of the plate.After this, place the reaction plate in a thermal cycler, and follow the procedure outlined in the text protocol. To perform the reverse transcription reaction, prepare the reaction mix in a centrifuge tube according to the text protocol. Then, add 15 microliters of the mix to each well of the plate containing ligation product.Mix the contents of the plate on a shaker at 1, 900 rpm for one minute, followed by a brief centrifuge of the plate. Next, place the plate in a thermal cycler, and follow the procedure outlined in the text protocol. Then, prepare the miR-Amp reaction mix, and add 45 microliters of the mix to each well of a new reaction plate.After this, add five microliters of the RT product to each well of the reaction plate. Mix the plate on a shaker at 1, 900 rpm for one minute, followed by a brief centrifuge of the plate. Then, place the plate in a thermal cycler, and follow the procedure outlined in table one of the text protocol.First, dilute each cDNA sample in TE buffer. Then, prepare the PCR reaction mix in a centrifuge tube. adding 10%of the volume in excess.Transfer 19 microliters of the reaction mix to each well of the plate. Seal the plate, and centrifuge it briefly. Finally, perform the thermal protocol on an RT-PCR system, as outlined in table two of the text protocol.Serial dilutions of spike-in control were performed in order to choose which was the best concentration to add in all plasma samples of this particular case series. Thanks to this protocol, it was possible to evaluate all the biomarkers in every single patient, as well as a single biomarker in the overall case series. In this protocol, a panel of angiogenesis-related circulating miRNAs in relation to progression-free survival, overall survival, and objective response rate was analyzed in patients with colorectal cancer treated with a chemotherapy regimen.Comparing the expression levels of miRNA between baseline and first clinical evaluation, an increase in hsa-miR-155-5p was observed. This increase was associated with shorter PFS and OS.While attempting this procedure, it's important to remember to perform the steps using phenol-chloroform under a fume hood. Following this procedure, other methods like isolation and analysis of circulating proteins or coding RNAs can be performed in order to answer additional questions, like evaluation of a more exhaustive panel of circulating biomarkers.After its development, this technique paved the way for researchers in the field of liquid biopsy to explore the expression of circulating biomarkers in cancer patients. Don't forget that working with blood and chemical reagents can be extremely hazardous, and precautions such as gloves and laboratory coat should always be taken while performing this procedure.

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Research

JoVE Journal

Cancer Research

5.3K Görüntüleme.  Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS. Bu yöntem, çeviriaraştırma alanındaki anahtar soruların yanıtlanmasına yardımcı olabilir, örneğin dolaşan biyobelirteçler ile hasta prognozu arasındaki ilişki gibi. Bu tekniğin en büyük avantajı, düşük miktarda giriş materyali ile kolay ve güvenilir bir metodoloji ile dolaşan biyobelirteçlerden oluşan bir panelin ekspresyonunu değerlendirme şansıdır. Bu tekniğin etkileri kanser hastalarının tedavisine kadar uzanır, çünkü hastaların karar verme ve tedavi...