**2. Methodological challenges in miRNA profiling design**

### **2.1. Sample collection**

repetitive sequences resulting in structural mutations (deletion, duplication and translocation) during tumor development [5]. Regulation of miRNA expression is an important mechanism by which tumor-suppressor proteins and oncogenic proteins exert some of their effects. A decrease or mutation in tumor-suppressor miRNAs can lead to overexpression of oncogenic proteins, in contrast to an overexpression of oncogenic miRNAs which can reduce expression of tumor suppressors. For example, tumor suppressor of the let-7 family targets RAS oncogene, which is involved in cell growth, differentiation and survival; reduced expression of let-7 miRNAs correlates with poor survival in many cancers [6]. In contrast, oncogenic miR-NAs in the cluster miR-17-92, comprising six miRNAs (miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1 and miR-92a-1), inhibits PTEN to activate AKT signaling and promote cancer cell

miRNAs have been considered promising candidates as diagnostic and prognostic biomarkers for the strong correlation between expression patterns of miRNAs and disease status and for the differences between normal and cancer tissues. This reflects the current evidence that some miRNAs are overexpressed or downregulated exclusively or preferentially in certain

Although miRNA profiling of tumors has been reported in solid tissues, obtained by invasive procedures, as an excellent prognostic test [8], routine biopsies from any organ for miRNA profiling are not practical options. Different studies suggest that circulating miRNAs are reliable indicators of pathological change because of their stability and protection against RNase digestion resulting from the formation of complexes with specific protein or incorporation within circulating exosomes or other extracellular vesicles (EV) [9]. In this context, accumulating evidence suggests that tumor cells are able to alter the function of both local and distant normal cells, thereby promoting tumor growth and metastasis, through the transfer of EV cargo [10]. miR-21, which targets the tumor-suppressor gene PTEN and programmed cell death 4 (PDCD4), is one of the first discovered and most investigated circulating miRNAs. Its upregulation seems to be of diagnostic and prognostic value in a variety of solid and hematological malignancies. High serum levels of miR-21 were strongly associated with lymph node metastasis, advanced-stage clinical disease and poor survival [11]. In patients with ovarian cancer, high serum levels of miR-34a were associated with lymph node disease and distant

Also, in patients with prostate cancer, plasma levels of miR-21, miR-141 and miR-221 were significantly higher in patients with metastases as compared to patients with localized or

Currently, a variety of miRNA detection methods, including northern blotting, in situ hybridization, quantitative reverse transcription PCR (qRT-PCR), microarray and deep sequencing, are commonly used [14]. However, miRNA profiling in biofluid samples is affected by a range of pre-analytical and analytical challenges in experimental design, from sample collection to profiling and data analysis (**Figure 1**). In this chapter, we will propose a workflow for exosomal miRNA detection from sera samples using the Affymetrix GeneChip microarray platform as a powerful molecular approach for biomarker discovery to translate into clinical practice.

survival [7].

32 Biomarker - Indicator of Abnormal Physiological Process

cancer types.

metastases [12].

locally advanced-stage disease [13].

Blood sample processing has a substantial impact on the results of miRNA profiling. During blood collection, it is important to avoid cellular contamination and hemolysis that can occur during phlebotomy as miRNAs derived from red and white blood cells risk to mask the intensities of truly circulating miRNA species. Residual platelets and microparticles can also affect the miRNA profile so an additional centrifugation is recommended prior to freezing samples. Moreover, biofluidics contain inhibitors of the reverse transcriptase and polymerase enzymes that can inhibit the enzymatic reactions in RT-qPCR so it is important to minimize the carryover of inhibitors into the RNA.

In our protocol, blood samples were collected and processed according to the national cancer institute (NCI's) Early Detection Research Network (EDRN) standard operating procedures for the collection and preparation of serum [15].

Whole blood samples were collected in red-top vacutainer tubes. Blood samples were incubated at room temperature for 30 min to allow complete coagulation. Coagulated samples were then centrifuged at 1500 × *g* for 20 min at room temperature to separate serum. The serum was transferred to new cryotubes with care so as to not to disturb the red blood layer and then centrifuged for 5 min at 3000 × *g* to remove cells. Aliquots of 1.5 ml of supernatant containing the cell-free serum were stored in cryotubes at −80°C until RNA extraction. Hemolyzed samples were excluded from further analysis.

#### **2.2. Exosome isolation and characterization**

The established standard for exosome isolation is ultracentrifugation [16]. However, this method cannot discriminate between exosomes and other microvesicles because different vesicles of similar size as well as protein aggregates can co-sediment at 100,000 × *g*.

Recently, methods claiming fast and simple exosome-purification procedures without ultracentrifugation are commercially available by various firms that use polymer-based precipitation or immune capture by antibody-coated beads.

However, according to the International Society for Extracellular Vesicles (ISEV) the separation of non-vesicular entities, such as protein complexes, from EV is not fully achievable by common EV isolation protocols, including centrifugation protocols or commercial kits. On the other hand, a list of EV-specific markers that distinguish subsets of EVs from each other is not proposed [17]. The ISEV provides minimal requirements to claim the presence of EV in pellets isolated from different methods. They suggested assessing the protein composition in at least a semi-quantitative manner in any EV preparation (Western blots, flow cytometry or mass spectrometry techniques). Size distribution of EVs, such as nanoparticle-tracking analysis (NTA), dynamic light scattering or resistive pulse sensing, needs to be analyzed. However, the values acquired with these techniques should be compared with TEM, AFM or other microscopy techniques, since they do not distinguish membrane vesicles from co-isolated non-membranous particles of similar size.

In our laboratory, serum exosomes underwent isolation by miRCURYTM Exosome Isolation Kit (Exiqon, Vedbaek, Denmark). The process is based on capturing water molecules which otherwise form the hydrate envelope of particles in suspension. The samples (1.5 ml) thawed on ice or at 4°C, were mixed with a precipitation solution in order to reduce the hydration of the particles. This allows precipitation of the subcellular particles below 100 nm with a lowspeed centrifugation step after incubation at 4°C for 1 h. The last pellet, containing exosomes, was resuspended using 240 μL of the provided resuspension buffer and used for further RNA extraction or stored at −20°C.

NTA was performed using a NanoSight LM10-HS microscope equipped with NTA software v3.1 (NanoSight Ltd., UK). Background was extracted and the automatic setting for minimum expected particle size, minimum track length and blur settings was employed. Each sample was diluted at 1:10,000 in sterile-filtered PBS (Sigma, USA). A video of typically 60 s duration was made, with a frame rate of 30 frames per second, and particle movement was analyzed by NTA software. Only measurements with >1000 completed tracks were analyzed (**Figure 2**).

We also have used 20 μL of human CD63 coated beads (Thermo Fisher Scientific, Inc) per 50 μL sample in order to isolate CD63 positive sub-populations of exosomes from total exosome isolations derived from serum samples. The isolated exosomes were stained for typical exosome markers, such as CD63, and CD9 was analyzed with the Attune NxT Flow Cytometer (Thermo Fisher Scientific, Inc.) (**Figure 3**).

Isopropanol was added to the supernatant and the solution was loaded onto a spin column. Thus, only molecules of RNA will bind to the resin, while proteins will be removed in the flow through. The RNA bound to the column was then washed with provided wash solution in order to remove any remaining impurities and then eluted with 100 μL of RNase

**Figure 2.** Extracellular vesicle NanoSight data. Example NanoSight NTA video frame (A), NanoSight NTA particle size/

Circulating MicroRNA Profiling in Cancer Biomarker Discovery

http://dx.doi.org/10.5772/intechopen.75981

35

concentration for 3 different miRCURYTM Exosome Isolation Kit recovered exosome pellets (B).

We also evaluated miRNA recovery from serum samples by the use of the exogenous synthetic miRNA cel-mir-39 (Applied Biosystems, Inc.) as standard control. For this procedure during RNA isolation, a known amount of cel-mir-39 (25 fmol) was spiked in each serum sample after the denaturation step. Consistent and homogeneous miRNA recovery leads to cel-miR-39 levels that are similar in all samples. A standard curve was generated using five ten-fold dilution of cel-miR-39 (1.4 × 10–4–1.4 × 10–8 ng) processed in parallel with qRT-PCR of experimental samples. The copy number of spiked cel-miR-39 was estimated, plotting Ct values, versus the copy number of the synthetic miRNA. The recovery of the synthetic miRNA is considered consistent if cel-miR-39 Ct values are within the range of acceptability (≤2 ×

Exosomal RNA purity was assessed by Nanodrop ND-1000 UV spectrometer (Thermo Scientific, Wilmington, DE) at the absorbance of 230, 260 and 280 nm. RNA samples extracted

free water.

standard deviation) in replicate isolations.

**2.4. RNA quantification and quality control**

#### **2.3. RNA purification**

RNA extraction method is another important issue in miRNA profiling as the exosomal RNA pattern is highly correlated with the sample source. The principles for isolating miRNA for profiling are, in general, the same for isolation of total RNA, except that miRNA isolation protocols are adapted to retain the small RNA fraction. In different studies describing exosome RNA, a number of alternative RNA extraction methods have been used, including phenolbased techniques, combined phenol and column-based approaches and pure column-based techniques [18–22].

For RNA purification from exosomes, we used the miRCURYTM RNA Isolation Kit (Exiqon, Vedbaek, Denmark) that is based on spin column chromatography using a proprietary resin as the separation matrix. First, membrane particles and cells were lysed using the lysis solution and then proteins were precipitated using the provided protein precipitation solution.

**Figure 2.** Extracellular vesicle NanoSight data. Example NanoSight NTA video frame (A), NanoSight NTA particle size/ concentration for 3 different miRCURYTM Exosome Isolation Kit recovered exosome pellets (B).

Isopropanol was added to the supernatant and the solution was loaded onto a spin column. Thus, only molecules of RNA will bind to the resin, while proteins will be removed in the flow through. The RNA bound to the column was then washed with provided wash solution in order to remove any remaining impurities and then eluted with 100 μL of RNase free water.

We also evaluated miRNA recovery from serum samples by the use of the exogenous synthetic miRNA cel-mir-39 (Applied Biosystems, Inc.) as standard control. For this procedure during RNA isolation, a known amount of cel-mir-39 (25 fmol) was spiked in each serum sample after the denaturation step. Consistent and homogeneous miRNA recovery leads to cel-miR-39 levels that are similar in all samples. A standard curve was generated using five ten-fold dilution of cel-miR-39 (1.4 × 10–4–1.4 × 10–8 ng) processed in parallel with qRT-PCR of experimental samples. The copy number of spiked cel-miR-39 was estimated, plotting Ct values, versus the copy number of the synthetic miRNA. The recovery of the synthetic miRNA is considered consistent if cel-miR-39 Ct values are within the range of acceptability (≤2 × standard deviation) in replicate isolations.

#### **2.4. RNA quantification and quality control**

common EV isolation protocols, including centrifugation protocols or commercial kits. On the other hand, a list of EV-specific markers that distinguish subsets of EVs from each other is not proposed [17]. The ISEV provides minimal requirements to claim the presence of EV in pellets isolated from different methods. They suggested assessing the protein composition in at least a semi-quantitative manner in any EV preparation (Western blots, flow cytometry or mass spectrometry techniques). Size distribution of EVs, such as nanoparticle-tracking analysis (NTA), dynamic light scattering or resistive pulse sensing, needs to be analyzed. However, the values acquired with these techniques should be compared with TEM, AFM or other microscopy techniques, since they do not distinguish membrane vesicles from co-isolated

In our laboratory, serum exosomes underwent isolation by miRCURYTM Exosome Isolation Kit (Exiqon, Vedbaek, Denmark). The process is based on capturing water molecules which otherwise form the hydrate envelope of particles in suspension. The samples (1.5 ml) thawed on ice or at 4°C, were mixed with a precipitation solution in order to reduce the hydration of the particles. This allows precipitation of the subcellular particles below 100 nm with a lowspeed centrifugation step after incubation at 4°C for 1 h. The last pellet, containing exosomes, was resuspended using 240 μL of the provided resuspension buffer and used for further RNA

NTA was performed using a NanoSight LM10-HS microscope equipped with NTA software v3.1 (NanoSight Ltd., UK). Background was extracted and the automatic setting for minimum expected particle size, minimum track length and blur settings was employed. Each sample was diluted at 1:10,000 in sterile-filtered PBS (Sigma, USA). A video of typically 60 s duration was made, with a frame rate of 30 frames per second, and particle movement was analyzed by NTA software. Only measurements with >1000 completed tracks were analyzed (**Figure 2**). We also have used 20 μL of human CD63 coated beads (Thermo Fisher Scientific, Inc) per 50 μL sample in order to isolate CD63 positive sub-populations of exosomes from total exosome isolations derived from serum samples. The isolated exosomes were stained for typical exosome markers, such as CD63, and CD9 was analyzed with the Attune NxT Flow Cytometer (Thermo

RNA extraction method is another important issue in miRNA profiling as the exosomal RNA pattern is highly correlated with the sample source. The principles for isolating miRNA for profiling are, in general, the same for isolation of total RNA, except that miRNA isolation protocols are adapted to retain the small RNA fraction. In different studies describing exosome RNA, a number of alternative RNA extraction methods have been used, including phenolbased techniques, combined phenol and column-based approaches and pure column-based

For RNA purification from exosomes, we used the miRCURYTM RNA Isolation Kit (Exiqon, Vedbaek, Denmark) that is based on spin column chromatography using a proprietary resin as the separation matrix. First, membrane particles and cells were lysed using the lysis solution and then proteins were precipitated using the provided protein precipitation solution.

non-membranous particles of similar size.

34 Biomarker - Indicator of Abnormal Physiological Process

extraction or stored at −20°C.

Fisher Scientific, Inc.) (**Figure 3**).

**2.3. RNA purification**

techniques [18–22].

Exosomal RNA purity was assessed by Nanodrop ND-1000 UV spectrometer (Thermo Scientific, Wilmington, DE) at the absorbance of 230, 260 and 280 nm. RNA samples extracted using the miRCURYTM method produced RNA concentrations above the lower detection limit of 2 ng/μL of the NanoDrop instrument and less than 10 ng/μL. Moreover, the miRCURYTM RNA isolation method showed a high average A260/280 ratio (1.9 ± 0.1) but low average

A260/230 ratio (<2.0). These values are consistent and acceptable in samples containing very

Circulating MicroRNA Profiling in Cancer Biomarker Discovery

http://dx.doi.org/10.5772/intechopen.75981

37

Because of the absence of 18S and 28S rRNA species in serum, the quality of isolated RNA samples might not be assessable by means of RNA integrity number (RIN), as a rule for total RNA samples to use for microarray procedures. On this basis, we used the Agilent Small RNA

miRNA expression profiling has allowed the identification of miRNAs that are involved in many biological processes, including organism development and establishment and maintenance of tissue differentiation [23, 24]. Thus, miRNAs are being explored as elements for cell-fate reprogramming in stem-cell applications or as biomarkers for identifying the origin of cancers of unknown primary sites. The expression pattern of miRNAs is widely different being tissue specific and related to developmental stages. Measuring miRNA expression can also be useful for system-level studies of gene regulation, especially when miRNA profiles are integrated with mRNA profiling. Circulating extracellular miRNAs, including exosome miRNA, are quantifiable in a range of specimen types including serum, plasma, urine and formalin-fixed tissue block. Hence, they are important as non-invasive biomarkers for many molecular diagnostic applications, including cancer [25, 26], cardiovascular and autoimmune diseases [27] and forensics [28]. **Table 1** shows new and promising miRNAs as potential bio-

The yield of RNA extracted from biofluids is usually very low, in the order of 1–10 ng. If we consider that miRNAs represent only 0.01% of total RNA, the strategies used for their detection and quantification are crucial. Some general consideration must be made in high-throughput miRNA profiling. First, the length of mature miRNA (19–25 nucleotides) is too low to allow annealing to the traditional primers during the reverse transcription step. Second, unlike mRNAs, miRNAs lack poly(A) tail, a region frequently used to anneal complementary and universal primers for RNA enrichment or reverse transcription. Third, miRNAs exist in different isoforms, so-called isomiRs, that are functional and evolutionarily important and, inside the same family (e.g., the let-7 family), can differ by a single base to the reference miRNA sequence. Depending on the goals of an miRNA profiling experiment, measurement of different forms may be required, even if the large majority of miRNAs typically show only modestlength heterogeneity. Another challenge for miRNA high-throughput profiling is variance in miRNA GC content that is reflected in different melting temperatures (Tm) of annealing reactions. To date, three major approaches are used for miRNA profiling: quantitative reverse transcription PCR (qRT-PCR), hybridization-based methods (microarrays) and next-generation sequencing (NGS) (RNA-seq). Microarrays were among the first hybridization-based methods

assays that are able to resolve fragments in the range 0–150 nucleotides (**Figure 4**).

low amounts of RNA, as biofluid samples.

**3. MiRNA profiling and data analysis**

markers for diagnosis and prognosis of different cancers.

to be used for parallel analysis of large numbers of miRNAs.

**3.1. General consideration for high-throughput miRNA profiling**

**Figure 3.** Flow analysis on exosome sub-populations (A). Exosomes isolated with CD63 showed that serum-derived exosome in our samples express high level of CD63 marker (82.6%) and a moderate level of CD9 markers (32.8%) (B).

**Figure 4.** Bioanalyzer analysis of serum exosomal small RNA. Exosome total RNA derived from serum was analyzed using Small RNA Kit in an Agilent 2100 Bioanalyzer. The electropherograms show the size distribution in nucleotides (nt) and fluorescence intensity (FU) of ladder (A) and exosome total RNA (B). The peak at 4 nt is an internal standard. The Small RNA region is visible in the interval of 0–150 nucleotides, including the miRNAs in the sizes between 10 and 40 nucleotides.

A260/230 ratio (<2.0). These values are consistent and acceptable in samples containing very low amounts of RNA, as biofluid samples.

Because of the absence of 18S and 28S rRNA species in serum, the quality of isolated RNA samples might not be assessable by means of RNA integrity number (RIN), as a rule for total RNA samples to use for microarray procedures. On this basis, we used the Agilent Small RNA assays that are able to resolve fragments in the range 0–150 nucleotides (**Figure 4**).
