METHOD FOR ISOLATING MICRORNAS (MIRNAS) AND THEIR USE

Description

1 / 21 METHOD FOR ISOLATING MICRORNAS (miRNAs) AND THEIR USE FIELD OF APPLICATION

[0001] The present invention applies to the field of biotechnology, focusing on use in laboratory settings, and discloses a method for isolating microRNAs (miRNAs) from biological samples of biofluids, such as saliva, plasma, blood, serum, among others, by means of the lyophilization technique and their use as biomarkers for early diagnosis and prognosis of diseases, as well as responsiveness to drugs and chemotherapeutic agents. FUNDAMENTALS OF THE INVENTION

[0002] miRNAs are RNAs that regulate mRNA gene expression, being responsible for numerous biochemical processes, including being recognized as inducers of diseases such as cancer and inflammatory processes, and are therefore used as biomarkers for drug treatment and diagnostic markers for diseases.

[0003] Exosomal localization of miRNAs requires extracellular vesicle (EV) isolation processes prior to miRNA extraction. The gold standard method for vesicle isolation in biofluids is ultracentrifugation. Furthermore, studies propose using EVs themselves as carriers in the production of more specific drugs for each pathology. However, new methods have been introduced, such as EV precipitation reagents, like those in commercial kits (exoEasy Maxi Kit, exoRNeasy Midi and Maxi Kits, miRCURY Exome Kits, among others), filter columns, size exclusion columns, and lipid-nano probe systems. These techniques present additional costs and, moreover, none have been Petition 870240111518, dated 12 / 31 / 2024, page 9 / 37 2 / 21 adequately described for saliva samples, and some are not indicated for nucleic acid analysis.

[0004] The methodology for isolating miRNAs from saliva is poorly described and, to date, no study has demonstrated isolation by replacing ultracentrifugation with lyophilization during the process, with high purity and concentration of miRNAs.

[0005] Thus, in order to solve the problems presented above, the present invention discloses a method for isolating microRNAs (miRNAs) from saliva samples using the lyophilization technique.

[0006] The method of the present invention promotes greater purity, speed and identification of circulating miRNAs originating from saliva. Until now, the isolation of salivary miRNA has not been described using this technique, which is important for diagnostic tests and the production of drugs with biomarkers and carriers. In addition, the equipment used is cheaper, ensuring greater financial accessibility, and the transport of samples does not require refrigeration. STATE OF THE ART

[0007] The scientific article “OPTIMIZING PRESERVATION OF EXTRACELLULAR VESICULAR miRNAs DERIVED FROM CLINICAL CEREBROSPINAL FLUID” describes the detection of miRNAs in extracellular vesicles (EVs) isolated from blood, cerebrospinal fluid (CSF), and other biofluids from patients with glioblastoma.

[0008] However, in the document above, miRNAs were not isolated from saliva, and the isolation method presents steps distinct from the method of the present invention. Petition 870240111518, dated 12 / 31 / 2024, page 10 / 37 3 / 21 as well as the process for obtaining cDNA. Another difference is the lyophilization time achieved by the present invention, which is much shorter and with distinct objectives. The document above aimed at sample preservation, while the present invention aims at sample concentration to obtain extracellular vesicles and isolate microRNAs.

[0009] The scientific article “SALIVARY MICRORNAS AND ORAL CANCER DETECTION” reveals the detection of microRNAs (miRNAs) in human saliva, using a molecular assay (qPCR Taqman) for the diagnosis of oral cancer.

[0010] However, the saliva used to obtain miRNAs in the article above was not lyophilized in the process, and the isolation method has different steps, and the techniques for preparing cDNA and analyzing microRNA are completely different from those of the present invention.

[0011] Patent application WO2016165831 aims to provide a method for the lyophilization of RNA for application in pharmaceutical compositions, vaccines and diagnostic kits.

[0012] Unlike the present invention, in the above document the RNA was isolated and then lyophilized to be used as a reagent in products such as vaccines and kits, which distinguishes it from the present invention, in which the saliva sample was lyophilized and subsequently the total RNA was isolated and the microRNA analyzed.

[0013] Patent application WO2009143181 discloses a method for diagnosing oral cancer in patients. The method includes the steps of: (a) detecting microRNA (miRNA) in a sample of the patient's saliva and (b) determining whether the Petition 870240111518, dated 12 / 31 / 2024, page 11 / 37 4 / 21 level is increased or decreased when compared with a standard control, thus providing a diagnosis of oral cancer. In some modalities, step (a) comprises an amplification reaction, for example, a polymerase chain reaction (PCR), especially a reverse transcription RT-PCR.

[0014] However, the saliva samples for obtaining miRNAs in the document above were not lyophilized in the process, and the isolation method also has different steps from the method of the present invention. SUMMARY OF THE INVENTION

[0015] miRNAs are RNAs that regulate mRNA gene expression, being responsible for numerous biochemical processes, including being recognized as inducers of diseases such as cancer, and are therefore used as biomarkers for drug treatment and diagnostic markers for diseases. The methodology for isolating miRNAs from saliva is poorly described and, to date, no study has demonstrated isolation by replacing ultracentrifugation with lyophilization during the process, with high purity and concentration of miRNAs.

[0016] In view of this context, the present invention discloses a method for isolating microRNAs (miRNAs) from biological samples of biofluids, such as saliva, plasma, blood, serum, among others, by means of the lyophilization technique and their use as biomarkers, promoting greater purity, speed and identification of circulating miRNAs originating from saliva or other biofluids. BRIEF DESCRIPTION OF THE FIGURES Petition 870240111518, dated 12 / 31 / 2024, page 12 / 37 5 / 21

[0017] The present invention may be better understood through the brief description of the following figures:

[0018] Figures 1A-1D reveal the analysis of ultracentrifuged (Figure 1A) and lyophilized (Figure 1B) salivary vesicles (Bars: 200 nm), the QELS (quasi-elastic light scattering) analysis of extracellular vesicles in suspension comparing ultracentrifugation and lyophilization (Figure 1C), and the Dot Blotting analysis demonstrates specific proteins of vesicles labeled with CD9 and CD63 antibodies (Figure 1D).

[0019] Figure 2 shows a graph demonstrating a higher concentration (ng / pL) of total RNA obtained by lyophilization compared to ultracentrifugation (p < 0.001).

[0020] Figures 3A-3D show graphs indicating that there is no difference between the expression of microRNAs isolated by lyophilization and by ultracentrifugation. miR-16 (Figure 3A), miR-21 (Figure 3B), miR-33a (Figure 3C), and miR-146b (Figure 3D) were used. DETAILED DESCRIPTION OF THE INVENTION

[0021] The invention can be better understood through the following detailed description, in accordance with the attached figures.

[0022] The present invention describes a method for isolating extracellular vesicles from salivary samples using the lyophilization technique followed by isolation of microRNAs (miRNAs) and their use as biomarkers for early diagnosis and prognosis of diseases, as well as responsiveness to drugs and chemotherapeutic agents.

[0023] The method for isolating microRNAs (miRNAs) of the present invention can be carried out from a biological sample, preferably a biofluid, in which vesicles Petition 870240111518, dated 12 / 31 / 2024, page 13 / 37 6 / 21 extracellular fluids (EVs) are isolated from the biological sample by a lyophilization process, and then miRNAs are isolated from the lyophilized EVs. The miRNA isolation method of the present invention comprises the steps of: (a) Collect a biological sample; (b) Process the biological sample; (c) Isolate extracellular vesicles (EVs) from the processed sample; and (d) Isolate miRNAs from the EVs.

[0024] The collection of the biological sample (step a) according to the present invention is described in more detail in the following Examples. The biological sample can be selected from the group comprising saliva, blood, serum, plasma, lymph, interstitial fluid, urine, among other biofluids, preferably saliva. Saliva is collected by rinsing with 5 ml of physiological saline and can be stored in a freezer between -40 and -80°C until processing.

[0025] The processing of the biological sample (step b) comprises centrifuging, transferring the supernatant containing the VEs to a new tube / container, discarding the pellet, centrifuging again and, finally, if necessary, freezing the material for the next step (isolation). Ideally, the separation of the pellet from the supernatant should be performed within two hours of collection, thus preventing degradation of the material. In a modality where blood is used, the material must be centrifuged within 2 hours of collection.

[0026] The isolation of EVs (step c) from the processed biological sample (product of step b) is carried out by Petition 870240111518, dated 12 / 31 / 2024, page 14 / 37 7 / 21 Lyophilization means, which can be carried out in a drying chamber, in a benchtop lyophilizer flask, or in any container or apparatus suitable for the lyophilization process. After a freezing period of 18 to 24 hours at a temperature between -40°C and -80°C, the supernatant containing the VEs can be transferred to the drying chamber or benchtop lyophilizer flask. The process progresses at a rate of ± 0.8 mm per hour, being approximately 14 hours (overnight) for the samples studied. After complete lyophilization, if not used immediately, the samples are stored at -80°C or at room temperature for up to 15 days, until the characterization of the VEs (step c1) and isolation of the microRNA (step d). The lyophilization product according to the present invention is described in more detail in the following Examples.

[0027] After step (c), the characterization of the VEs (step c1) that were isolated from the processed biological sample can also be performed. The characterization of the VEs comprises measuring the size and density of the VEs (step c1.1); performing electron microscopy (step c1.2); analyzing by Dot Blotting (step c1.3), or combinations thereof. For step c1, any known VE characterization method in the state of the art can be additionally applied, such as Western Blotting, flow cytometry, NTA (Nanoparticle Tracking Analysis), dot blotting, or ELISA.

[0028] In step c1.1, the size and density of the VEs can be measured by dynamic light scattering. Lyophilized samples can be resuspended in 150 to 200 µl of PBS. The effective diameter distribution of the VEs can Petition 870240111518, dated 12 / 31 / 2024, page 15 / 37 8 / 21 can be measured in a particle size and zeta potential analyzer at 20 to 25°C using 4 to 5 μL of sample in 1 to 2 µL of saline solution. A non-negative constraint least squares (NNLS) algorithm based on the light scattering intensity of each particle can generate multimodal particle size diameter distributions.

[0029] In step c1.2, electron microscopy characterization can be performed with preparations labeled with contrast agents, such as uranyl acetate. For this, lyophilized samples are resuspended in 150 to 200 µL of PBS. Samples can be deposited on copper or nickel grids coated with synthetic thermoplastic resins, selected from a group comprising polyvinyl formaldehyde (e.g., Formvar, carbon), where 10–20 µL of the samples are pipetted onto the coated grids and allowed to adhere for 2–2.5 h at room temperature (20–30°C). Then the grids can be fixed by inversion in a drop of 4% formaldehyde in PBS pH 7.2 for 30–40 min, washed in water, and stained in a mixture of 3% methylcellulose and 4% uranyl acetate (9:1) for 10–10 minutes on ice. Microvesicle contrast can be performed using 5% uranyl acetate for 30 seconds at room temperature.

[0030] Alternatively, the fixed microvesicles can be blocked in blocking buffer (150 mM glycine or ammonium chloride in PBS pH 7.2) for 10 to 20 min, followed by incubation in PBS pH 8.0 containing 2% BSA and 0.01% saponin for 30 to 40 min at room temperature. Then, the grids can be incubated with commercial anti-CD9 and anti-CD63 mouse antibodies diluted in PBS pH 8.0 containing Petition 870240111518, dated 12 / 31 / 2024, page 16 / 37 9 / 21 Samples can be incubated in 2% BSA and 0.01% saponin (1:100) for 120 to 180 min at room temperature (20-30°C). Then, samples can be washed in PBS pH 8.0 containing 2% BSA and 0.01% saponin, incubated with 10 nm colloidal gold-conjugated goat anti-mouse IgG (at a minimum dilution of 1:100) in blocking buffer for 60 to 90 min, and washed in water by inversion in a drop (100 pL). Subsequently, samples can be refixed in 1% glutaraldehyde in PBS for 5 to 7 min, washed in water, and stained in a mixture of 3% methylcellulose and 4% uranyl acetate (9:1) for 10 min by inversion in a drop (50-100 μL) kept on ice. The grids are dried on filter paper and stored at room temperature. The samples can be observed using a transmission electron microscope operating at 80 kV.

[0031] In step cl.3, samples can also be analyzed by Dot Blotting, whereby 25-50 pg of VEs from ultracentrifuged (control) or lyophilized (present invention) samples can be applied to the nitrocellulose membrane. The membrane can be blocked with TBS supplemented with 3-5% skim milk and 0.01% to 0.05% non-ionic surfactants, selected from a group comprising polysorbate, whose purpose is to prevent protein adsorption and promote blocking, such as Tween 20, Tween 80, for 60 to 120 min, followed by incubation with primary antibodies, mouse anti-CD9 or anti-CD63 antibody at 0.2 pg / mL to 1 pg / mL (1:1000) using the same blocking buffer) for 60 to 90 min. After washing by inversion in a drop (50 to 100 µL) and incubation with peroxidase-conjugated anti-mouse secondary antibodies (1:1500; diluted in blocking buffer; 100-200 ng / mL), the reactive areas Petition 870240111518, dated 12 / 31 / 2024, page 17 / 37 10 / 21 can be visualized using horseradish substrate conjugated to luminol (20 µg to 100 µg; 500 μL to 1000 μL)... Protein concentration can be determined by the Bradford, Lowry, DC Protein Assay, or BCA assay, using the V fraction of albumin as a standard. The characterization of VEs is described in more detail in the following Examples.

[0032] The isolation of miRNAs (step d) from lyophilized samples containing EVs can be performed by two consecutive purifications.

[0033] The first purification of lyophilized samples can be carried out by adding suitable reagents, agitation, incubation, homogenization, and centrifugation, in order to obtain a sample with three phases, namely: an upper (aqueous) phase containing RNA; an intermediate phase containing DNA; and a lower phase containing reagents and proteins. The aqueous phase containing RNA is collected and transferred to a new container / tube, which undergoes similar steps of reagent addition, homogenization, incubation, and centrifugation, then the supernatant is discarded, and the pellet is retained and dried as much as possible. The resulting purified pellet containing RNA is then homogenized and quantified in the spectrophotometer. The remaining material from the intermediate phase and the phase containing reagents and proteins can optionally be used for DNA and protein extraction, or the material can simply be discarded.The initial purification can be performed using manual techniques, such as the one exemplified above, or commercial kits, which are mechanical techniques in which the sample is placed and the microRNA is obtained. Petition 870240111518, dated 12 / 31 / 2024, p. 18 / 37 11 / 21

[0034] The second purification comprises steps similar to the first purification, where the centrifugation step for discarding the supernatant can be repeated two to four times before drying the resulting pellet. At the end of the purifications, the pellet (containing RNA) is resuspended and the total RNA concentration and purity can be analyzed by spectrophotometry. The second purification can also be performed using commercial kits, but is preferably performed as described below.

[0035] The first purification is performed in order to obtain microRNA from the lyophilized sample, while the second purification is performed in order to improve the quality and purity of the isolated sample (miRNA) from lyophilized EV samples. The experimental details of each purification are detailed in the following Examples.

[0036] The method of the present invention may further comprise a step of analyzing the miRNAs (step e) isolated in the previous step d. The miRNAs can be analyzed, for example, by means of the RT-PCR technique (reverse transcriptase reaction followed by polymerase chain reaction), from the preparation of complementary DNA (cDNA) of the pre-defined miRNA sequences and, subsequently, applying PCR using an RT-PCR instrument. Other miRNA analysis techniques known in the prior art can be applied, such as PRC array, microArray, with confirmation of results by the RT-PCR technique. This step is described in more detail in the following Examples.

[0037] Therefore, the miRNA isolation method of the present invention may more broadly comprise the following steps: Petition 870240111518, dated 12 / 31 / 2024, page 19 / 37 12 / 21 (a) Collect a biological sample; (b) Process the biological sample; (c) Isolate extracellular vesicles (EVs) from the processed sample; (c1) Characterize the VEs; (d) Isolate miRNAs from EVs; and (e) Analyze the miRNAs. EXAMPLES Example 1: Collection of saliva samples

[0038] In step (a), saliva samples were collected after the patient rinsed their mouth with 5 mL of physiological solution for one minute, collecting approximately 1 to 3 mL of sample per patient and, after collection, the samples were stored at -20°C until processing.

[0039] Patients were instructed to undergo pre-treatment, which includes performing oral hygiene 1 hour before collection and abstaining from food, cigarettes and alcoholic beverages for at least 1 hour before collection. Example 2: Processing of the biological sample

[0040] In step (b), after thawing at room temperature (20 to 30°C), and centrifugation in a range of 1,500 g to 10,000 g for 10 to 15 min at a temperature of 20 to 30°C, the supernatant containing the VEs was transferred to a new 15 mL tube with the pellet discarded, followed by a new centrifugation between 12,000 g and 20,000 g for 20 to 30 min at a temperature of 20 to 30°C to remove cellular debris. Finally, the material was frozen at -80°C with the tube held in a vertical position. Example 3: Isolation of extracellular vesicles (EVs) Petition 870240111518, dated 12 / 31 / 2024, p. 20 / 37 13 / 21

[0041] Test group (lyophilization): after 24 h of freezing, the supernatant was transferred to the drying chamber of the benchtop lyophilizer (LT1000, Enterprise II, Terroni Equipamentos LTDA). The process progresses at a rate of ± 0.8 mm per hour, being approximately 14 h (overnight) for the samples studied. After complete lyophilization, the samples were stored at -80°C until the moment of VE characterization and microRNA isolation.

[0042] Control group (centrifugation): samples were thawed at room temperature (20 to 30°C) and ultracentrifuged at 160,000g for 2 hours at 4°C, rotor 70 Ti (Beckman Coulter Ultracentrifuge, USA). Immediately, the samples were transferred to 1.5 mL tubes, and the pellet was subsequently resuspended in 100 μL of PBS for measurement of extracellular vesicle size and density, dot blotting analysis, and electron microscopy. To obtain total RNA, 1 mL of Trizol reagent was added to the pellet. Example 4: Characterization of extracellular vesicles (EVs) A) Measurement of endoplasmic reticulum (EV) size and density by quasi-elastic light scattering (QELS)

[0043] The lyophilized samples were resuspended in 200 mL of PBS. The effective diameter distribution of the VEs was measured on the NanoBrook Omni, a particle size and zeta potential analyzer, at 25°C using 5 μL of sample in 1 mL of saline solution. A non-negative constraint least squares (NNLS) algorithm based on the light scattering intensity of each particle generated multimodal particle size diameter distributions. Petition 870240111518, dated 12 / 31 / 2024, page 21 / 37 14 / 21

[0044] Figure 1C reveals the QELS analysis of extracellular vesicles in suspension comparing lyophilization (gray column) and ultracentrifugation (black column), demonstrating high density of EVs divided into two main populations for the two groups: (30-70 nm and 340-370 nm). B) Electron microscopy - preparations labeled with uranyl acetate

[0045] The lyophilized samples were resuspended in 200 pL of PBS. Samples were deposited on grids coated with polyvinyl formaldehyde and allowed to adhere for 2 h at room temperature (20 to 30°C). Then the grids were fixed by inversion in a drop of 4% formaldehyde in PBS for 30 min, stained with 3% methylcellulose and 4% uranyl acetate (9:1) for 10 minutes on ice. Alternatively, the fixed microvesicles were blocked in 150 mM glycine in PBS pH 7.2 for 10 min, followed by incubation in PBS pH 8.0 blocking containing 2% BSA and 0.01% saponin for 30 min. Then, the grids were incubated with mouse anti-CD9 and anti-CD63 antibodies (0.2 pg / mL; 1:100; Thermo Fisher Scientific, Waltham, Massachusetts, USA) in blocking buffer for 3 h at room temperature (20 to 30°C). Then, the samples were washed in blocking buffer and incubated with 10 nm colloidal gold-conjugated goat anti-mouse IgG (1:100, in blocking buffer) for 1 h and washed in PBS by inversion in a drop (50 to 100 µL). Subsequently, the samples were refixed in 1% glutaraldehyde in PBS for 5 min, washed in water, and stained in a 3% methylcellulose and 4% uranyl acetate solution (9:1) for 10 min by inversion in a drop (50 to 100 µL) on ice. The samples were observed in Petition 870240111518, dated 12 / 31 / 2024, page 22 / 37 15 / 21 JEOL 1200 transmission electron microscope operating at 80 kV as described by Pereira et al., 2018.

[0046] The results of this analysis can be seen in Figure 1A, where the EVs were ultracentrifuged, resuspended in saline solution and contrasted with uranyl acetate / methylcellulose (inset in A, arrow shows CD63 localization in membrane - bar: 100 nm). In Figure 1B, the EVs were lyophilized, resuspended in saline solution and stained with uranyl acetate / methylcellulose (inset in B demonstrates CD9 immunolocalization in membrane - bar: 100 nm). C) Dot Blotting Analysis

[0047] Twenty-five micrograms of EVs from control (ultracentrifugation) or lyophilized samples were applied to the nitrocellulose membrane. The membrane was blocked with TBS supplemented with 5% skim milk and 0.01% Tween was incubated for 1 h, followed by incubation with primary antibodies, mouse anti-CD9 or anti-CD63 antibody (1:1000 using the same blocking buffer) for 1 h. After washing and incubation with horseradish peroxidase-conjugated secondary antibodies (1:1500 diluted in blocking buffer), reactive spots were visualized using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, Massachusetts, USA) according to the manufacturer's instructions. Protein concentration was determined by the Bradford assay using the fraction V of albumin as standard (FIGURE 1D). EXAMPLE 5: Isolation of miRNAs

[0048] The first purification was performed by adding 1 to 1.5 mL of Trizol to the lyophilized sample, followed by Petition 870240111518, dated 12 / 31 / 2024, page 23 / 37 16 / 21 vortex for 50 to 60 seconds and incubate for approximately 8 minutes at a temperature between 15°C and 30°C in a 1.5 mL microtube. 200 to 250 µL of PA chloroform were added; followed by vigorous homogenization for 15 to 20 seconds using a vortex mixer or manually. Then, the samples were incubated at 15°C to 30°C for 2 to 3 minutes and centrifuged at 12,000 g for 15 to 20 minutes at a temperature between 2°C and 8°C, resulting in three phases in the sample: an upper (aqueous) phase containing RNA; an intermediate phase containing DNA; and a lower phase with reagents and proteins. The entire aqueous phase (200 to 700 µL), containing RNA (including miRNAs), was collected and transferred to a new tube. Next, 500 to 600 µL of chilled PA isopropanol was added to the new tube containing RNA, gently homogenized for approximately 1 min, followed by incubation at 15°C to 30°C for 10 to 15 minutes, then centrifugation at 12,000 g for 10 to 12 minutes at a temperature between 2°C and 8°C, and finally, the supernatant was discarded.Next, the RNA-containing pellet was thoroughly dried for 5 to 10 minutes, and 20 to 30 pL of RNase-free water was added. Then, the purified RNA-containing material was homogenized and quantified using a spectrophotometer. Measurements were performed using the sample solvent, which is RNase-free water, for equipment calibration. Then, 1 to 2 pL of the sample was loaded onto the equipment's base pedestal, and the reading was taken. This reading provided the 260 / 280 ratio, the 260 / 230 ratio, and the concentration of the samples.

[0049] The second purification of the isolated samples was performed with the addition of 400 to 500 μL of phenol solution. Petition 870240111518, dated 12 / 31 / 2024, page 24 / 37 17 / 21 PA / chloroform PA 1:1 was added to the RNA isolated in the first purification, followed by gentle homogenization for 2 to 2.5 minutes, centrifugation at 12,000 g for 15 to 20 minutes at 4 to 8°C, followed by collection and discarding of the vesicles formed on the sample surface. Subsequently, 900 µL to 1.25 mL of 95% PA ethanol maintained at room temperature (20 to 30°C) and 40 to 50 µL of 3 M sodium acetate (pH 5.2) were added, and the mixture was gently homogenized for 2 to 2.5 minutes, followed by incubation in a -80°C freezer for 30 to 40 minutes, centrifugation at 10,000 g for 20 to 25 minutes at a temperature of 2°C to 8°C. Next, in order to remove any remaining reagents (washing the material), the supernatant was discarded and 1 to 1.2 mL of 70% ethanol was added, followed by centrifugation at 7,500g for 5 to 8 minutes at a temperature of 2°C to 8°C. The supernatant was then discarded, and this step was repeated twice. The tube was inverted onto absorbent paper for 10 to 15 minutes to allow the pellet to dry.

[0050] Next, the pellet was resuspended in 20 to 30 μL of RNase-free water, and the total RNA concentration was analyzed using a microvolume spectrophotometer for use / application in nucleic acids, cell culture, and customized methods. Purity was assessed using the spectrophotometer at absorbances of 230, 260, and 280 nm. A 260 / 280 ratio of 1.80 and a 260 / 230 ratio of 2.0 were considered the ideal standard for the samples tested. Measurements were performed using the sample solvent, which was RNase-free water for equipment calibration, and then 1 to 2 μL of the sample was loaded onto the equipment's base pedestal and the reading was taken. This reading Petition 870240111518, dated 12 / 31 / 2024, page 25 / 37 18 / 21 provided the 260 / 280 ratio, the 260 / 230 ratio, and the total RNA concentration of the samples.

[0051] The purifications for the control samples (ultracentrifuged) were performed in the same way as for the samples of the present invention (lyophilized). After ultracentrifugation, the supernatant was discarded, the pellet was transferred to a new 1.5 mL tube, and 1 to 1.5 mL of Trizol was added to the tube containing the sample, followed by vortexing for 50 to 60 seconds and incubation for approximately 5 to 8 minutes at a temperature between 15°C and 30°C in a 1.5 mL microtube. The process was followed as described in Example 5. Example 6: Analysis of miRNAs by RT-PCR

[0052] In step (e), for the preparation of complementary DNA (cDNA), the reverse transcriptase (RT) reaction was performed using a commercial kit, such as TaqMan™ MicroRNA. Reverse Transcription Kit®, TaqMan Advanced miRNA Assays (Thermo Fisher) and miRCURY LNA RT Kit (Qiagem). Total RNA (10 ng to 30 ng), 1X stem-loop RT primer (3 pL) (mir-21, mir-33a, mir-146b) and endogenous control (mir-16), 3.33 U / pl of reverse transcriptase (1 pL), 0.25 U / pl of RNase inhibitor (0.19 pL), 0.25 mM dNTPs (0.15 pL), and 1X buffer (1.50 pL) were used in a final volume of 15 pL, and incubated at 16°C for 30 min, 42°C for 30 min and 85°C for 5 min, in the MyCycler™ thermocycler (BioArt, USA), according to the TaqMan®MicroRNA Assays protocol.

[0053] Negative samples were included for Reverse transcriptase (no RT, i.e., samples that have passed through the thermocycler with all reagents, but without the reverse transcriptase enzyme), as well as blank samples (without Petition 870240111518, dated 12 / 31 / 2024, pp. 26 / 37 19 / 21 samples, but with RT primer) as negative controls. After cDNA preparation from miRNA via reverse transcriptase (RT), 1.33 μL of RT reaction (cDNA) was combined with 1.0 μL of TaqMan MicroRNA Assay (20X probe) and 10 μL of TaqMan® Universal PCR Master Mix II, No UNG (PN). 4428175) in a final reaction volume of 20 μl. PCR was performed using the Agilent Technologies Stratagene Mx3005P with a cycle of 95°C for 10 minutes (first segment), followed by 95°C for 15 seconds and 60°C for 60 seconds, for a total of 40 cycles (second segment). Each TaqMan Assay was run in triplicate (n=3). The C(q) value is considered for evaluating the expression between the methods. Example 7: Sample preparation protocol

[0054] It is worth noting that each group used for the sample preparation protocol was intended to observe the behavior of the sample with different treatments in relation to purity and concentration.

[0055] The samples were subjected to several preparation protocols prior to the isolation of total RNA, rich in miRNAs, as no information on the use of the TRzol Reagent was found in the manufacturer's protocol. The reagent for extracting total RNA from biofluids (TRIzol™ Reagent User Guide, 2016) was used, but no information was found in the literature regarding the lyophilization of biofluids to obtain RNA. In more detail, the supernatant consists of total RNA, to which the isopropanol reagent was basically used to precipitate the total RNA, which contains miRNAs. When Trizol was added to the lyophilized endocrine systems (ESSs), they were digested (broken down), exposing the microRNA. Therefore, when collecting the aqueous phase, RNA including microRNAs is collected. Petition 870240111518, dated 12 / 31 / 2024, page 27 / 37 20 / 21 microRNAs. However, the use of Trizol alone in the initial purification would ultimately isolate any RNA, given its non-specificity. Therefore, the remaining steps were fundamental for the isolation of miRNA in the present invention. Based on this, five types of sample preparation protocols were performed to evaluate whether modifying the total RNA isolation technique could affect concentration, purity, etc. These protocols are described below: 1) Isolation by the Trizol method with the lyophilized (dried) sample (No Treatment + Trizol Group); 2) Addition of 200 μL of RNase-free water to a 15 lyophilized sample followed by isolation using the Trizol method (H2O + Trizol group); 3) Filtration of the salivary supernatant (0.2 µm filter) prior to lyophilization and isolation by the Trizol method (Filter + Trizol group); 4) Addition of 200 pL of RNase-free water to the lyophilized sample followed by isolation using the Trizol method and freezing at -80°C for 7 days, followed by isolation using the Trizol method (H2O + (-80°C) + Trizol group); and 5) Lyophilization combined with sample storage for 15 days at room temperature (Group 15 days + Trizol).

[0056] For the 5 sample preparation protocols (lyophilization) performed and the control group (ultracentrifugation), the isolation of exosomal RNA, using the Trizol method, was based on the manufacturer's protocol for mRNA extraction (Trizol™ Reagent User Guide, 2016). The total RNA concentration was analyzed using the NanoDrop® Spectrophotometer 2000 (Thermo Scientific, Petition 870240111518, dated 12 / 31 / 2024, page 28 / 37 21 / 21 Wilmington, DE, USA). At least three independent experiments were performed for each group (n=3).

[0057] In the protocols tested, a higher concentration (ng / μΐ) of total RNA was obtained by lyophilization compared to ultracentrifugation (p < 0.001), thus, the lyophilization method has a greater capacity to isolate the EVs as a whole in relation to ultracentrifugation (Figure 2). RESULTS

[0058] All groups demonstrated high purity, both lyophilized and ultracentrifuged samples. qRT-PCR analysis demonstrated the presence of miR-16 (Figure 3A), miR-21 (Figure 3B), miR-33a (Figure 3C) and miR-146b (Figure 3D) in lyophilized and ultracentrifuged samples. The results showed, by qRT-PCR, the amplification of some miRNAs (mir-16, mir-21, mir-33a and mir-146b) in the supernatant of lyophilized and ultracentrifuged saliva, characterizing lyophilization as a new method for isolating extracellular vesicles from biofluids.

[0059] The present invention has been disclosed in this descriptive report in terms of its preferred embodiment. However, other modifications and variations are possible from the present description, and are still within the scope of the invention disclosed herein. Petition 870240111518, dated 12 / 31 / 2024, p. 29 / 37

Claims

1 / 4 CLAIMS 1. A method for isolating microRNAs (miRNAs) from a biological sample, CHARACTERIZED in that the miRNAs are located inside extracellular vesicles (EVs), the method comprising the steps of: (a) Collecting a biological sample; (b) Processing the biological sample; (c) Isolating the extracellular vesicles (EVs) from the biological sample; (d) Isolating the miRNAs from the EVs.

2. Isolation method, according to claim 1, CHARACTERIZED in that the biological sample comprises saliva, blood, serum, plasma, lymph, interstitial fluid, urine, among other biofluids, preferably saliva.

3. Isolation method, according to claim 1 or 2, CHARACTERIZED in that the collection (step a) is carried out after rinsing the individual in question with 5 mL of physiological solution for 1 minute, thus collecting 1 to 3 mL of the biological sample, wherein the biological sample is stored between -40 and -80°C until step b.

4. Isolation method, according to any one of claims 1 to 3, CHARACTERIZED in that the sample processing (step b) comprises centrifuging, transferring the supernatant containing the VEs to a new tube or container, discarding the pellet, centrifuging again and, optionally, freezing the material for the next step (isolation - step c).

5. Isolation method, according to claim 4, CHARACTERIZED in that the sample is thawed at room temperature (20 to 30°C), and centrifuged in a range of 1,500 g to 10,000 g for 10 to 15 min at a temperature of 20 to 30°C, the supernatant containing the VEs is transferred to a new 15 mL tube with the pellet discarded, followed by a new centrifugation between 12,000 g and 20,000 g for 20 to 30 min at a temperature of 20 to 30°C to remove cellular debris.

6. Isolation method, according to any one of claims 1 to 5, CHARACTERIZED in that the isolation of EVs (step c) is carried out by lyophilization, wherein, after lyophilization, the sample can be stored again at -80°C or at room temperature, until step C (isolation of EVs).

7. Isolation method, according to claim 6, CHARACTERIZED in that during lyophilization (step c), the frozen supernatant is transferred to the drying chamber of the lyophilizer, where the process progresses at a rate of ± 0.8 mm per hour.

8. Isolation method, according to claim 6 or 7, CHARACTERIZED in that, optionally, after lyophilization (step c), the VEs are characterized (step c1) by quasi-elastic light scattering (QELS), or electron microscopy, or Dot Blotting analysis, or combinations thereof.

9. Isolation method, according to any one of claims 1 to 8, CHARACTERIZED in that the isolation of miRNAs (step d) is carried out by means of at least two purification steps of the lyophilized sample. Petition 870240111518, dated 12 / 31 / 2024, page 31 / 37 3 / 4 10. Isolation method, according to claim 9, CHARACTERIZED in that the first purification comprises the addition of reagents, agitation, incubation, homogenization and centrifugation; wherein a sample with three phases is obtained, the first being the upper or aqueous phase containing RNA; the second the intermediate phase containing DNA; and the third the lower phase containing reagents and proteins; wherein the aqueous phase is dried to obtain pellets containing miRNA.

11. Isolation method, according to claim 9 or 10, CHARACTERIZED in that the reagents used in the first purification are PA chloroform, PA isopropanol or commercial reagents.

12. Isolation method, according to any one of claims 9 to 11, CHARACTERIZED in that the second purification comprises adding reagents and centrifuging the aqueous phase of the first purification two to four times before drying the pellet.

13. Isolation method, according to any one of claims 9 to 12, CHARACTERIZED in that the reagents used in the second purification are PA phenol / PA chloroform solution, PA ethanol, 3 M sodium acetate and 70% ethanol.

14. Isolation method, according to claim 1, CHARACTERIZED in that the method optionally comprises the analysis of miRNAs (step e) isolated in step d by means of reverse transcriptase reaction followed by PCR (RT-PCR), wherein: a complementary DNA (cDNA) is made from the isolated miRNAs; and Petition 870240111518, dated 12 / 31 / 2024, page 32 / 37 4 / 4 the miRNAs are analyzed by means of RT-PCR.

15. Use of isolated miRNAs, according to the method defined in any of claims 1 to 14, CHARACTERIZED by the fact that it is in the preparation of biomarkers for early diagnosis and prognosis of diseases related to the miRNA in question.

16. Use of isolated miRNAs, according to claim 15, CHARACTERIZED by the fact that it is used in the evaluation of responsiveness to chemotherapeutic drugs related to the miRNA in question. Petition 870240111518, dated 12 / 31 / 2024, pp. 33 / 37

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