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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Cancer Research

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

Published: March 14th, 2019

DOI:

10.3791/58615

1Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, 2Department of Medical Oncology, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, 3Unit of Biostatistics and Clinical Trials, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS

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 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' and the ligation of an adapter at 5' 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.

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 hav....

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NOTE: Perform all of the following steps under a sterilized fume hood according to Good Laboratory Practice (GLP).

1. Plasma Collection and Storage

  1. Collect 3 mL of peripheral blood sample in a K3E EDTA tube.
  2. Centrifuge the tube at room temperature at 1,880 x g for 15 min.
  3. Recover the supernatant plasma in aliquots of 450 µL.
  4. Store the samples at -80 °C until use.
    NOTE: Process the peripheral b.......

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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's instructions and with.......

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miRNAs are small non-coding RNAs (18-25 nucleotides in length) capable of binding the 3' 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,.......

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

....

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Name Company Catalog Number Comments
Rnase-free Safe-lock 1.5 mL tubes Eppendorf 0030 123.328
Rnase-free Safe-lock 2 mL tubes Eppendorf 0030 123.344
Rnase-free 20 µL tips Starlab S1123-1810
Rnase-free 200 µL tips Starlab S1120-8810
Rnase-free 1000 µL tips Starlab S1122-1830
mirVana PARIS RNA and Native Protein Purification Kit Thermo Fisher AM1556
TaqMan Advanced miRNA cDNA Synthesis Kit Thermo Fisher A28007
100% ethanol anidrous ACS grade Carlo Erba Reagents 414605
2-mercapto-ethanol Sigma-Aldrich M3148
TaqMan Fast Advanced Master Mix Thermo Fisher 4444558
TaqMan Advanced miRNA Assays Thermo Fisher A25576
7500 Real-Time PCR System Applied Biosystems 4406984
0.2-2, 1-10, 2-20, 20-200, 100-1000 µL laboratory pipettes
Benchtop microcentrifuge
Vortex
Benchtop heating block
Fume hood
0.2 mL PCR tubes

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