Methods for isolating circulating nucleic acids from urine samples

Abstract

Provided herein is a method for characterizing target cell-free nucleic acid (cfNA) molecules present in a biological sample, such as a urine sample. This includes isolating total cfNA from a biological sample without pretreatment, such as centrifugation to remove cellular debris, and characterizing the target cfNA molecule based on the isolated total cfNA. If the target cfNA is a low molecular weight (LMW) molecule, the method further includes a fractionation step to obtain LMW nucleic acid from the total cfNA before characterization. This method can detect significantly more copies of the target cfNA molecule compared to existing methods that generally discard cellular debris from biological samples. Another method is also provided that substantially recovers cfNA from normally discarded cellular debris, which can also detect significantly more copies of the target cfNA molecule.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 223,542, filed July 19, 2021, the disclosure of which is incorporated herein by reference in its entirety. [Background technology]

[0002] The presence of circulating nucleic acids (NAs), e.g., circulating DNA (e.g., cell-free DNA, i.e., cfDNA) or circulating RNA (e.g., cell-free RNA, i.e., cfRNA) in urine is well established. For example, in pregnant women, extracellular fetal DNA has been shown to be present in the maternal circulation and detectable not only in maternal blood but also in maternal urine samples, although at much shorter lengths and at much lower concentrations (Chan et al. 2003; Tsui et al. 2012; Botezatu et al. 2000; Al-Yatama et al. 2001; Majer et al. 2007; Li et al. 2003; Illanes et al. 2006; Koide et al. 2005; Shekhtman et al. 2009). These findings clearly limit the potential clinical utility of urinary DNA, as previous studies have suggested. Interestingly, most of the cited studies isolated urinary cfDNA from maternal urine samples and used centrifugation to remove cell pellets as a pretreatment step before DNA isolation, similar to existing approaches to recover cfNA from urine samples. Studies have shown that circulating fetal genetic material can be used to reliably determine fetal loci that are completely absent in the maternal genome, such as by PCR (polymerase chain reaction) techniques, with an example of such a fetal locus being a fetal Y chromosome-specific sequence that has been successfully identified in maternal urine (Tsui et al. 2012; Al-Yatama et al. 2001; Lin et al. Diagnostics 2021. 11(4)).Our studies also showed that liver-derived hepatitis B virus (HBV) DNA, hepatocellular carcinoma DNA biomarkers, and colorectal cancer (CRC) DNA biomarkers were detected in urine samples from patients with hepatitis B virus infection (Lin et al. Hepatology communication 2022; Jain et al. 2018), HCC (Lin et al. Diagnostics 2021. 11(8); Hann et al. 2017; Zhang et al. 2018; Kim et al. 2022), and CRC (Botezatu et al. 2000; Su et al. 2004; Su et al. 2005; Song et al. 2012), respectively.

[0003] Currently, almost all existing approaches, including commercial kits for cfNA recovery from urine samples, rely on pretreatment prior to DNA isolation, e.g., brief centrifugation at 1,000 rpm for 10 min or faster to remove cellular debris to increase DNA isolation efficiency. However, these existing cfDNA recovery approaches suffer from low recovery rates, even if the isolation methods are suitable for blood cfNA isolation (Chan et al. 2003; Tsui et al. 2012). Summary of the Invention

[0004] To address the aforementioned problems associated with existing cfNA detection approaches, the present disclosure provides methods for characterizing target cell-free nucleic acid (cfNA) molecules present in biological samples, such as urine samples, which can be quite challenging.

[0005] One such method provided herein essentially comprises the following two steps:

[0006] (1) isolating total cell-free nucleic acid from a biological sample without pretreatment of the biological sample to remove cellular debris (e.g., by centrifugation); and

[0007] (2) characterizing the target cell-free nucleic acid molecules from the isolated total cell-free nucleic acid obtained in step (1).

[0008] As used herein, the method is capable of detecting at least two times, and preferably at least six times, copies of a target cell-free nucleic acid molecule in a biological sample compared to when cellular debris is removed by pretreatment in the isolation step (1).

[0009] When the target cfNA molecule is low molecular weight (LMW) DNA, i.e., has a length shorter than about 1 kb, in order to increase the detection sensitivity, the method further comprises, after isolating the total cell-free DNA from the urine sample, obtaining low molecular weight DNA from the isolated total cell-free DNA before characterizing the target cell-free DNA molecule from the isolated total cell-free DNA. Correspondingly, characterizing the target cell-free DNA molecule from the isolated total cell-free DNA comprises characterizing the target cell-free DNA molecule from the low molecular weight DNA.

[0010] Another such method provided herein comprises three main steps:

[0011] (a) a step of pretreating a biological sample, specifically comprising the steps of: centrifuging the biological sample to obtain a supernatant fraction and a pellet fraction; washing the pellet fraction with a washing solution to obtain a washed-out fraction; and combining the supernatant fraction and the washed-out fraction to obtain a composite fraction;

[0012] (b) isolating total cell-free nucleic acid from the complex fraction obtained from step (a); and

[0013] (c) characterizing the target cell-free nucleic acid molecules from the isolated total cell-free nucleic acid obtained from step (b).

[0014] As used herein, the method is capable of detecting at least 1.25 times more copies of a target cell-free nucleic acid molecule from a biological sample compared to when total cell-free nucleic acid is isolated solely from the supernatant fraction of said pre-treated biological sample.

[0015] Details will be provided below. [Brief description of the drawings]

[0016] [Figure 1] Figure 1 shows the association of transrenal Y-Chr DNA with cell pellets after centrifugation. Briefly, 10 mL of urine from two different collections of urine from two donors (donor 1 and donor 2) of pregnant mothers with male fetuses was spiked with 1x105 copies / mL of synthetic double-stranded DNA, artificial spike-in (SPKN), respectively, centrifuged at 1,500 rpm and 4°C for 10 min, and the supernatant and pellet were fractionated. DNA was isolated, eluted, and quantified by qPCR assay for spike control SPKN and Y-Chr (Lin et al. Diagnostics 2021. 11(4)). The percentage recovery from SPKN and the total recovery from Y-Chr were calculated and plotted. [Diagram 2] Figure 2 shows the association of transrenal HBV DNA with the cell pellet after centrifugation. Briefly, 10 mL of urine from different collections from four donors (donors 1-3) with chronic HBV infection was spiked with 1x105 copies / mL of synthetic double-stranded DNA, SPKN, and centrifuged at 1,500 rpm for 10 min at 4°C, and the supernatant and pellet were fractionated. DNA was isolated, eluted, and quantified by qPCR assays of spiked control SPKN and HBV pol / S assays as detailed in Table 1. Percent recovery from SPKN input and total HBV DNA recovery were calculated and plotted. [Diagram 3]Figure 3 demonstrates that preferential isolation of low MW urinary DNA from total urinary DNA using gel electrophoresis improved the sensitivity of detection of CRC-associated K-ras mutations. Briefly, total and low MW urinary DNA were prepared as described in the text and subjected to RE-PCR assay for mutant K-ras DNA. The photograph shows the difference in RE-PCR results between total and low MW urinary DNA from six different individuals diagnosed with colorectal cancer. A doubling of detection sensitivity in low MW DNA was demonstrated. [Figure 4] Figure 4 outlines the experimental procedure for obtaining cfDNA from urine samples of hepatocellular carcinoma (HCC) patients and investigating the detection sensitivity of two HCC-associated DNA markers, aberrant methylation of the RASSF1A gene (mRASSF1A) and hTERT-124 hotspot mutations (mTERT), using different methods. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] In a first aspect, the present disclosure provides a first method for characterizing a target cell-free nucleic acid molecule present in a biological sample. The first method essentially comprises the following two steps:

[0018] (1) isolating total cell-free nucleic acid from a biological sample without pretreatment of the biological sample to remove cellular debris from the biological sample; and

[0019] (2) characterizing the target cell-free nucleic acid molecules from the isolated total cell-free nucleic acid obtained in step (1).

[0020] As used herein, the method is capable of detecting at least twice as many copies of a target cell-free nucleic acid molecule in a biological sample as compared to when cellular debris is removed by pretreatment in the isolation step (1).

[0021] As used herein and throughout this disclosure, the term "biological sample" refers to a sample obtained from one or more biological subjects, including one or a combination of a urine sample, a serum sample, a plasma sample, a saliva sample, a sweat sample, or a lymphatic fluid sample. These biological samples contain nucleic acids but generally do not contain cells. Such biological samples can be obtained from a single source (e.g., a urine sample) or from a combination of multiple sources, such as a combination of a urine sample and a plasma sample. Such biological samples can be obtained from a single subject (e.g., a cancer patient or a pregnant woman) or can be pooled samples from multiple subjects. Such biological samples can be obtained fresh, thawed from a frozen sample, or processed biological samples, so long as such processing does not remove cellular debris therefrom.

[0022] As used herein, the term "nucleic acid" refers to DNA and / or RNA molecule(s), and terms such as "cell-free nucleic acid", "circulating nucleic acid", and the like refer to one or more nucleic acid molecules that exist or are present in one particular biological sample (e.g., a urine sample or a plasma sample), as defined above. For example, the cell-free nucleic acid present in a urine sample may include transrenal DNA (i.e., DNA originally originating from the circulation passes through renal filtration to become transrenal DNA), or may include apoptosis-derived nucleic acid molecules (i.e., derived from apoptotic cells from the urinary tract of the subject from whom the urine sample is obtained). In another example, the cell-free nucleic acid in a plasma sample may be derived from cell-free nucleic acid that coexists with other blood cells in the circulation. Here, the target cell-free nucleic acid molecule may be a DNA (e.g., cell-free DNA or cfDNA, etc.) molecule or an RNA molecule (e.g., microRNA or miRNA, cell-free RNA or cfRNA, etc.).

[0023] In the above isolation step (1), the phrase "pretreatment of biological sample to remove cellular debris therefrom" refers to a pretreatment step on biological sample or biological sample carried out before isolation step (1), which removes cellular debris contained in biological sample from biological sample. A typical example of such a pretreatment step includes a step of typically centrifuging biological sample (e.g., 1,500 RPM at 4°C for 10 minutes), which is conventionally applied in almost all existing target cell-free nucleic acid marker characterization methods for the purpose of providing a cleaner biological sample for the convenience and effectiveness of nucleic acid isolation and target marker detection. However, it should be noted that such a cell debris removal pretreatment step is not limited to centrifugation, and can also include other means or approaches.

[0024] In the above characterizing step (2), the terms "characterizing step," "characterize," "characterization," and the like are intended to include either one or both of qualitative (i.e., detecting or monitoring whether or not the cell-free nucleic acid molecule of interest is present in a biological sample) and quantitative (i.e., determining the level of copy number, weight, ratio, concentration, and the like, of whether or not the cell-free nucleic acid molecule of interest is present in a biological sample).

[0025] As used herein, the method is capable of detecting at least 2-fold, more preferably at least 6-fold, copies of a target cell-free nucleic acid molecule in a biological sample compared to when cellular debris is removed by pretreatment in the isolation step (1).

[0026] As shown in the examples below, when total cell-free nucleic acids are extracted directly from a biological sample (i.e., a urine sample), the ability to detect target cell-free DNA markers can be significantly increased (up to about 30-fold) compared to corresponding control experiments in which the biological sample was subjected to a conventional centrifugation pretreatment and only the supernatant fraction after centrifugation was used for DNA isolation and target marker detection.

[0027] For example, in Example 3 below, urine samples from pregnant mothers were tested for the ability to detect fetal Y-Chr DNA, demonstrating that the method without centrifugation (i.e., the "NC" group) dramatically detected 6.2-fold and 11.7-fold higher copies of such markers in urine samples compared to the conventional method (i.e., the "supernatant" group of the "centrifugation" group) (see Table 2).

[0028] In another example shown in Example 4 below, urine samples from hepatocellular carcinoma (HCC) patients were tested for the ability to detect HCC markers (i.e., methylated RASSF1A (mRASSF1A) and / or mutated hTERT (mTERT)), and it was uniquely demonstrated that the method not involving centrifugation (i.e., the "NC" group) dramatically detected 7.1-fold and 29.2-fold more copies of the mRASSF1A marker in urine samples compared to conventional methods (i.e., the "supernatant" group in the "centrifugation" group) (see Table 3), and with regard to the mTERT marker, at least 6-fold more copies of the marker could be found using the first method provided herein compared to conventional methods.

[0029] According to some embodiments of the method provided in this first aspect, the biological sample is a urine sample and the target cell-free nucleic acid molecule comprises at least one of a transrenal nucleic acid molecule or an apoptosis-derived nucleic acid molecule.

[0030] wherein optionally the target cell-free nucleic acid molecule is a target cell-free DNA molecule, and correspondingly the first method comprises the steps of:

[0031] Isolating total cell-free DNA from a urine sample; and

[0032] Characterizing the target cell-free DNA molecules from the isolated total cell-free DNA.

[0033] According to some embodiments, the target cell-free DNA molecules are low molecular weight (LMW) DNA, i.e., DNA having a length less than about 1 kb, and in order to increase the detection sensitivity, the method further comprises, after isolating the total cell-free DNA from the urine sample, obtaining low molecular weight DNA from the isolated total cell-free DNA before characterizing the target cell-free DNA molecules from the isolated total cell-free DNA. Correspondingly, characterizing the target cell-free DNA molecules from the isolated total cell-free DNA comprises characterizing the target cell-free DNA molecules from the low molecular weight DNA.

[0034] Herein, the step of obtaining low molecular weight DNA from the isolated total DNA can be carried out by a size differentiation approach selected from a carboxylated magnetic bead-based approach, an agarose gel-based chromatography approach, or a polyacrylamide gel-based chromatography approach.

[0035] In this specification, the step of characterizing the target cell-free DNA molecule from low molecular weight DNA can be realized by at least one of polymerase chain reaction (PCR) assay, sequencing assay, or hybridization assay. PCR assay includes normal PCR assay, real-time PCR assay, quantitative PCR assay, etc. Examples of sequencing assay include Sanger sequencing, next-generation sequencing. Examples of hybridization assay include Southern blot assay, microarray assay. These above-mentioned assays can further include the use of primers, probes, etc. with sequences specifically designed for target cell-free DNA.

[0036] As shown in Examples 2 and 4 below, according to some embodiments, the target cell-free DNA molecule is a cancer-associated DNA marker selected from mutated K-ras, methylated RASSF1A (mRASSF1A), or mutated TERT (mTERT).

[0037] As shown in Examples 1 and 3 below, according to some embodiments, a urine sample is obtained from a pregnant woman and the target cell-free DNA molecules are fetal DNA markers associated with gender, autosomal traits, or genetic diseases, where the fetal DNA markers can optionally include Y chromosome (Y-Chr) markers, where the autosomal traits can include fetal RhD status (e.g., in RhD-negative women) and the genetic diseases can include male-linked disorders, adrenal hyperplasia, myotonic dystrophy, achondroplasia, fetal aneuploidy, etc.

[0038] As shown in Example 1 below, according to some embodiments, the target cell-free DNA molecule comprises an HBV DNA marker. Optionally, the target cell-free DNA molecule may be a DNA marker for other viruses (e.g., HIV, HCV, Covid19, SARS, etc.) or other microorganisms, such as bacteria, fungi, etc.

[0039] Optionally, in the methods provided herein, the target cell-free nucleic acid molecule is a target cell-free RNA molecule, and correspondingly, the first method comprises:

[0040] isolating total cell-free RNA from the urine sample; and

[0041] Characterizing the target cell-free RNA molecules from the isolated total cell-free RNA.

[0042] According to some embodiments, the target cell-free RNA molecule comprises a microRNA.

[0043] As used herein, the step of characterizing the target cell-free DNA molecules from the low molecular weight DNA can optionally be performed by at least one of a reverse transcription polymerase chain reaction (RT-PCR) assay, an RNA sequencing assay, or a hybridization assay (e.g., a Northern blot or a microarray assay).

[0044] In any of the above method embodiments, the step of isolating total cell free nucleic acid from the biological sample can optionally be performed using a carrier RNA, such as tRNA.

[0045] In a second aspect, the present disclosure further provides a second method for characterizing target cell-free nucleic acid molecules in biological samples.Compared to the first method described above in the first aspect, the second method still includes a centrifugation pretreatment step to remove cell debris.More specifically, the method includes three main steps:

[0046] (a) pretreating a biological sample, specifically, centrifuging the biological sample to obtain a supernatant fraction and a pellet fraction; washing the pellet fraction with a washing solution to obtain a wash-off fraction; and combining the supernatant fraction and the wash-off fraction to obtain a composite fraction;

[0047] (b) isolating total cell-free nucleic acid from the complex fraction obtained from step (a); and

[0048] (c) characterizing the target cell-free nucleic acid molecules from the isolated total cell-free nucleic acid obtained from step (b).

[0049] As used herein, the method is capable of detecting at least 1.25-fold, and preferably at least 2-fold, copies of a target cell-free nucleic acid molecule from a biological sample as compared to isolating total cell-free nucleic acid solely from the supernatant fraction in a pretreated biological sample.

[0050] As shown in the Examples below, in urine samples in which the supernatant and pellet fractions were pre-processed by centrifugation before being subjected to DNA marker detection separately, the ability to detect target cell-free DNA markers can be increased (up to approximately 20-fold) when both fractions are combined for detection (i.e., the "combination approach") compared to control experiments in which only the supernatant fraction was examined (i.e., the "conventional approach").

[0051] For example, in Examples 1 and 3 below, it is demonstrated that the "combined approach" can detect 1.26 to 6.62 times more copies of the fetal Y-Chr DNA marker (see FIG. 1 and Table 2) and 1.34 to 1.98 times more copies of the HBV DNA marker (see FIG. 1 and Table 2) than the corresponding "conventional approach". In another example, in Example 4 below, it is independently demonstrated that the "combined approach" can detect 4.4 to 21.2 times more copies of the mRASSF1A marker (see Table 3) and at least 14.5 times more copies of the mTERT marker (see Table 3) than the corresponding "conventional approach".

[0052] In any embodiment of the method provided in this second aspect, the wash solution may have a pH of about 2.0-4.0 or may contain a salt (NaCl, KCl, MgCl, LiCl, sodium citrate, or any combination thereof) having a concentration of about 0.15-3 M.

[0053] A total of four specific examples are presented below, which are intended to further illustrate the invention disclosed in this specification and should not be construed as limiting the scope thereof in any way.

[0054] Working Example Example 1

[0055] Briefly, in this example, we use detectable male fetal DNA in maternal urine or HBV DNA from patients with chronic HBV infection as indicators of transrenal DNA to evaluate the impact of centrifugation-dependent pretreatment on cfDNA recovery.

[0056] 1. Materials and Methods

[0057] 1.1 Subjects and urine samples

[0058] For Y-Chr testing, archived urine samples collected on different dates from two third trimester donors carrying male infants were used in this study.

[0059] For HBV-DNA testing, each patient had an HBV serum viral load of at least 10 6 IU / mL serum and archived urine samples from three chronically HBV-infected donors who had detectable HBV DNA in their urine measured in a previous study were used.

[0060] 1.2 Urine sample collection

[0061] Briefly, 50 ml urine samples were collected in EDTA-containing urine storage tubes at two different time points with a 1-day interval. The urine sample collection method is detailed below. Specifically, to inhibit possible nuclease activity in the urine samples, freshly collected urine was immediately mixed with 0.5 mol / L EDTA (pH 8.0) to a final concentration of 10–50 mmol / L EDTA and stored at -70°C. To isolate total urinary DNA, frozen urine samples were thawed at room temperature and immediately placed in ice before DNA isolation. Thawed urine was processed for DNA isolation within 1 h.

[0062] 1.3 Urine sample pretreatment

[0063] First, 100 of synthetic double-stranded spike-in (SPKN) DNA fragments were added to the 15 ml urine sample collected above. 5 The urine samples were spiked at 100 copies / mL and pretreated by centrifugation prior to DNA isolation. Briefly, for pretreatment by centrifugation, urine samples were centrifuged at 1,500 RPM for 10 minutes at 4°C, and the supernatant was collected and separated from the cellular debris "pellet." DNA was isolated from each of the two urine fractions (supernatant and pellet).

[0064] 1.4 DNA isolation

[0065] DNA was isolated from each of the above urine fractions according to the total DNA isolation approach detailed below. Specifically, each urine sample was digested with proteinase K (1 mg / mL) in 2 M guanidine hydrochloride lysis buffer for 1 h. The urine lysate was then mixed with 0.8 volumes of 6 mol / L guanidine thiocyanate (Sigma, St. Louis, MO), 1 volume of isopropanol, 50 uL of MagsilRed (catalog number: A1641, Promega, Madison, WI) silica magnetic beads and incubated overnight at room temperature with gentle end-to-end mixing. The bead-DNA complexes were centrifuged, washed twice with 80% ethanol, and DNA was eluted with 20 μl of water.

[0066] 1.5 Real-time PCR quantification

[0067] Real-time PCR quantification of Y chromosome ("Chr") DNA using the Y-Chr qPCR assay (Lin et al. 11(4); Lin et al. Hepatology communication 2021) and HBV DNA using the HBV pol / s assay are detailed in Table 1. Sequences from the Y chromosome (Y-Chr) or HBV DNA were amplified on a Roche LightCycler480 instrument platform by real-time quantitative PCR to quantify the amount of isolated fetal DNA or HBV DNA in the different urine fractions. Each fraction was quantified in duplicate in 15 μl containing 6 μl of urine DNA sample using the Y chromosome DNA quantification kit (detailed in Table 1) and HBV pol / s DNA quantification kit (detailed in Table 1) according to the protocol. Synthetic spike-in SPKN DNA was quantified using the JBS artificial spike-in (SPKN) DNA quantification kit according to the manufacturer's specifications (detailed in Table 1) to estimate the copies recovered in each cfDNA sample. [Table 1]

[0068] The SPKN recovery from the input was calculated using equation (1): Total output / total input × 100% = SPKN% from input; (1)

[0069] The distribution of Y-Chr or HBV DNA in the supernatant or pellet fraction was calculated using equation (2): "Pellet" or "Supernatant" / (Pellet + Supernatant) × 100%; (2)

[0070] In the study with Y-Chr DNA shown in Figure 1, exogenous SPKN 131 bp dsDNA (SEQ ID NO: 7) was used as a control and the supernatant and cell pellet were fractionated by centrifugation. As expected, the top panel of Figure 1 shows that SPKN was largely recovered from the supernatant fraction, ranging from 48% to 99%, while the recovery of SPKN DNA from the pellet fraction was extremely low, ranging from 1% to 7.2%. Interestingly and unexpectedly, as shown in the bottom panel of Figure 1, the amount of Y-Chr DNA that could be recovered from the pellet fraction varied depending on the sample, but was usually significantly higher than the SPKN DNA control, ranging from 21% to 88% of the total Y-Chr detected.

[0071] This result shows that Y-Chr DNA has an unexpected association with cell debris pellet, unlike exogenously added SPKN DNA.This further suggests that the centrifugation pretreatment commonly used in almost all existing cfDNA isolation and detection methods loses a significant proportion of cfDNA from circulating or transrenal DNA by binding to cell debris in the pellet fraction after centrifugation.Therefore, by using the method described in the second aspect of the present disclosure above, which recovers cfDNA from the "pellet" fraction after centrifugation in addition to the "supernatant" fraction after normal centrifugation, the amount of cfDNA recovered is substantially significantly higher, and a significantly higher number of copies of Y-Chr DNA markers are detected. When the "pellet" fraction is combined with the "supernatant" fraction, the Y-Chr DNA markers are approximately 1.26-fold (i.e., 1 / 79.4%), 5.24-fold (i.e., 1 / 19.1%), 1.54-fold (i.e., 1 / 64.9%), 6.62-fold (i.e., 1 / 15.1%), 2.50-fold (i.e., 1 / 39.9%), and 1.72-fold (i.e., 1 / 58.2%) greater than the "supernatant" fraction alone in the six test groups shown in Figure 1.

[0072] In the study with HBV DNA shown in Figure 2, the exogenous spike 131 bp dsDNA (SEQ ID NO: 7) described above was also used as a control for the fractionation of the supernatant and cell pellet by centrifugation. As expected, as shown in the upper panel of Figure 2, SPKN was mostly recovered from the supernatant fraction, ranging from 77% to 82%, and only a small amount of SPKN DNA (1.5% to 21.4%) was recovered from the pellet fraction. Interestingly, and unexpectedly, but consistent with the results of Y-Chr DNA described above, a significant amount of HBV DNA was recovered from the cell debris pellet, ranging from 26% to 50% of the total HBV DNA detected, as shown in the lower panel of Figure 2. This indicates that a significant amount of HBV DNA may be associated with the cell debris pellet after the pretreatment step of centrifugation, which may have been lost in almost all existing cfDNA isolation and detection methods that only target the supernatant fraction.

[0073] Therefore, similar to the detection results of the fetal Y-Chr DNA marker shown in Figure 1, the combination of the "pellet" fraction with the conventional "supernatant" fraction is expected to detect approximately 1.84 times (i.e., 1 / 54.3%), 1.34 times (i.e., 1 / 74.5%), and 1.98 times (i.e., 1 / 50.4%) more HBV markers, respectively, for the three test groups shown in Figure 2, compared to the "supernatant" fraction alone.

[0074] Example 2

[0075] Removal of high MW DNA by gel electrophoresis facilitated the detection of mutant K-ras DNA in the urine of patients with colorectal disease.

[0076] To detect the codon 12 mutation of K-ras, restriction enzyme enrichment polymerase chain reaction (RE-PCR) was performed on total and low-MW urinary DNA as previously described (Su et al. 2004) (specific primers and assay conditions are shown in Table 1).

[0077] Briefly, total DNA or fractionated low MW DNA from 200 μL of urine was used for each assay. The PCR product by the RE-PCR assay was 87 bp, and the appearance of a 71 bp fragment after a second BstNI digestion is evidence of mutant K-ras DNA in the DNA sample. The detection limit of the RE-PCR assay is 15 copies of mutant K-ras per 100 ng of wild-type DNA per reaction (Su et al. 2004). As assay controls, DNA prepared from sources known to have mutant (human adenocarcinoma SW480 cells) or wild-type (human hepatoblastoma HepG2 cells) K-ras sequences was subjected to PCR. As expected, as shown in Figure 3, DNA prepared from HepG2 cells did not contain detectable levels of mutant K-ras (no 71 bp fragment appeared after a second BstNI digestion), whereas DNA prepared from SW480 cells contained mutant K-ras sequences (a 71 bp fragment was detected after a second BstNI digestion). For each urine sample that contained detectable mutant K-ras DNA, the presence of mutant K-ras sequences was more evident when low MW DNA was used in the assay compared with total urinary DNA, as shown by six different individuals in Figure 3 (i.e., “FX,” “GD,” “GG,” “GI,” “GM,” and “GN”).

[0078] To evaluate the improved detection sensitivity by LMW DNA fractionation, we performed end-point detection by two-fold serial dilution. Briefly, both total DNA and LMW DNA were first subjected to five two-fold serial dilutions (1:2, 1:4, 1:8, 1:16, and 1:32). Each dilution was subjected to RE-PCR assay to determine the dilution end point containing detectable K-ras mutations. The fold increase in low / total DNA was calculated and shown in Figure 3. The detection sensitivity increased in the range of 4-16-fold. This is quite unexpected, since the processing step of LMW DNA fractionation usually results in the loss of cfDNA from total DNA. As mentioned above, the K-ras RE-PCR assay is not a powerful assay, and its analytical sensitivity decreases about 10-20-fold with the addition of 100 ng of human wild-type background DNA (Su et al. 2004), so the removal of HMW DNA improved the detection sensitivity. The fold increase in mutant K-ras DNA detection in low MW (LMW) DNA per total urinary DNA was calculated and shown in Figure 3. Using low-MW urinary DNA as a substrate for K-ras mutation detection increased the detection sensitivity by 4- to 16-fold compared with using total urinary DNA, indicating that removal of high molecular weight (>1 kb) DNA by LMW DNA fractionation significantly improves the detection of K-ras mutations in urine.

[0079] Example 3

[0080] Briefly, in this example, we evaluate the impact of centrifugation-dependent pretreatment on cfDNA recovery using male fetal DNA detectable in maternal urine as an indication that relevant Y-Chr DNA may be washed out of the cell pellet.

[0081] 1. Materials and Methods

[0082] 1.1 Subjects and urine samples

[0083] The study used archived urine samples collected from one donor in the third trimester carrying a male fetus.

[0084] 1.2 Collection of urine samples

[0085] Briefly, 50 ml urine samples were collected in EDTA-containing urine storage tubes at two different time points with a 1-day interval. Urine sample collection is detailed below. Specifically, to inhibit possible nuclease activity in urine samples, freshly collected urine was immediately mixed with 0.5 mol / L EDTA (pH 8.0) to a final concentration of 10–50 mmol / L EDTA and stored at -70°C. To isolate total urinary DNA, frozen urine samples were thawed at room temperature and immediately placed in ice before DNA isolation. Thawed urine was processed for DNA isolation within 1 h.

[0086] 1.3 Urine sample pretreatment

[0087] 15 ml of the urine sample collected above was pretreated before DNA isolation, either by centrifugation or without centrifugation (hereafter abbreviated as "NC"). Briefly, in pretreatment with centrifugation, the urine sample was centrifuged at 1,500 RPM and 4 °C for 10 min, the supernatant was collected and isolated from the cell debris pellet (hereafter referred to as "pre-wash pellet" to distinguish it from the "post-wash pellet"). The supernatant was placed on ice, the pre-wash pellet was washed briefly with 1 ml of sodium citrate buffer (pH 3.0), the washing solution was collected by centrifugation at 13,000 RPM for 90 s, and three urine fractions (supernatant, wash-off, and post-wash pellet) were isolated from each urine fraction. In pretreatment without centrifugation (i.e., NC approach), DNA was isolated directly from the urine sample, and low-MW DNA fractionation was performed using carboxylated beads.

[0088] 1.4 DNA isolation

[0089] DNA was isolated from each of the above urine fractions or NC urine samples according to the total DNA isolation approach detailed below. Specifically, each urine sample was digested with proteinase K (1 mg / mL) in 2 M guanidine hydrochloride lysis buffer for 1 h. The urine lysate was then mixed with 0.8 volumes of 6 mol / L guanidine thiocyanate (Sigma, St. Louis, MO), 1 volume of isopropanol, 50 uL of MagsilRed (catalog number: A1641, Promega, Madison, WI) silica magnetic beads and incubated overnight at room temperature with gentle end-to-end mixing. The bead-DNA complexes were centrifuged, washed twice with 80% ethanol, and DNA was eluted with 20 μl of water.

[0090] 1.5 Real-time PCR quantification of Y chromosome ("Chr") DNA using the Y-Chr qPCR assay

[0091] Sequences derived from the Y chromosome (Y-Chr) were amplified by real-time quantitative PCR on a Roche LightCycler480 instrument platform to quantify the amount of isolated fetal DNA in the different urine fractions, as detailed in Table 1. Each fraction was quantified in duplicate in 15 μl containing 6 μl of urine DNA sample using the Y chromosome DNA quantification kit (JBS Science, Doylestown, PA) according to the protocol detailed in Table 1.

[0092] 2. Results

[0093] Table 2 summarizes the detection results for each fraction / sample (i.e., results regarding the ability to detect circulating cell-free fetal DNA from maternal urine samples using copies of the circulating cell-free fetal DNA marker "Y-Chr" as an indicator), specifically for each fraction / sample of the centrifugation pre-treated and non-centrifugation pre-treated urine samples (i.e., NC urine samples), specifically including the "supernatant," "wash removed," and "wash pellet" fractions, at each of the two different urine collection time points (i.e., collections #1 and #2).

[0094] As shown, the "supernatant" fraction contained approximately 7.99 and approximately 1.51 copies of detectable Y-Chr DNA (for collections #1 and #2, respectively), which essentially represents the amount of target DNA in urine samples detectable by traditional centrifugation-dependent approaches that typically only target the supernatant fraction after centrifugation.

[0095] Surprisingly and unexpectedly, when the pellet fraction, which is usually discarded after centrifugation, was washed, the resulting "washed off" fraction contained about 2.78 and 1.91 copies (for collections #1 and #2, respectively) of Y-Chr DNA, whereas the "washed pellet" fraction contained no detectable such target DNA. When the copies of Y-Chr DNA from the "supernatant" and "washed off" fractions were added together to determine the total copies of Y-Chr DNA recovered (i.e., "total recovered after centrifugation", 10.77 and 3.42 copies for collections #1 and #2, respectively), the recovered Y-Chr DNA was about 1.4-fold and 2.3-fold higher (for collections #1 and #2, respectively) than the "supernatant" alone. Thus, compared to conventional DNA isolation methods that only target the supernatant fraction after centrifugation, a surprising and significant proportion of the target Y-Chr DNA remains in the pellet fraction after centrifugation, which is usually discarded. It is estimated that the DNA obtained from the supernatant fraction after centrifugation and the DNA obtained from the pellet fraction after centrifugation combined account for approximately 25.8%–55.8% of the total target DNA that was otherwise lost (i.e., for collection #1: 2.78 / 10.77 = 25.8%, for collection #2: 1.91 / 3.42 = 55.8%).

[0096] Even more surprisingly and unexpectedly, when DNA was isolated directly from urine samples, completely omitting or removing the usual pretreatment step of centrifugation, the copies of target DNA detected thereby were dramatically higher than the copies of target Y-Chr DNA detected in the supernatant fraction after centrifugation. Specifically, the copy numbers of Y-Chr DNA detectable in the "no centrifugation" or "NC" group were 49.58 and 17.62 (for collections #1 and #2, respectively), which are 6.2-fold and 11.7-fold higher than those in the "supernatant" only group (for collections #1 and #2, respectively). Thus, compared to the centrifugation-free approach disclosed herein, it can be seen that 83.9% to 91.4% of the target Y-Chr DNA can be lost when DNA isolation is performed using a simplified conventional centrifugation method that only involves the supernatant fraction after centrifugation (i.e., for collection #1: (49.58-7.99) / 49.58=83.9%, and for collection #2: (17.62-1.51) / 17.62=91.4%). In other words, compared to conventional cfDNA isolation methods that require removal of cellular debris from urine samples (e.g., by centrifugation pretreatment), the method disclosed herein that substantially removes the cellular debris removal pretreatment can isolate approximately 5.2-10.7 times more detectable target Y-Chr DNA, thus dramatically improving the detection sensitivity and dramatically lowering the detection limit of such target low molecular weight (LMW) DNA in urine samples. [Table 2]

[0097] Example 4

[0098] Association of HCC-associated DNA markers with cell debris pellets.

[0099] Urine collected from patients with two known detectable HCC DNA markers in urine. As shown in Figure 4, different urine aliquots of 10 mL each were subjected to whole urine DNA isolation without centrifugation followed by fractionation to obtain low molecular weight (LMW) DNA shorter than 1 kb (i.e., "No Centrifugation" or NC group), or were subjected to centrifugation pretreatment and DNA isolation as above to obtain DNA from the supernatant (Sup) or pellet fractions (i.e., "Centrifugation" group). DNA was then subjected to methylated RASSF1A (mRASSF1A) and hTERT-124 mutation (i.e., mTERT) marker assays as detailed in Table 1. Summary results are shown in Table 3. [Table 3]

[0100] As shown in Table 3, the mRASSF1A marker was reproducibly detected in both urine fractions of the "centrifugation" and "no centrifugation" groups, although the amount was different. When the conventional cfDNA isolation method was applied (i.e., corresponding to the "supernatant" fraction of the "centrifugation" group), only 1.6 copies of the mRASSF1A marker were detected, whereas more copies of the marker were detected in the "pellet" fraction of the "centrifugation" group (32.3 copies in patient #1 and 5.4 copies in #2, respectively), similar to the Y-Chr observations shown in Table 2. Thus, when the "supernatant" and "pellet" fractions were combined, the total detectable levels of the mRASSF1A marker (i.e., "total recovery") were approximately 21.2- and 4.4-fold (patients #1 and #2, respectively) compared to the conventional method, which only targets the "supernatant" fraction after centrifugation. Moreover, when the assay was performed without centrifugation (i.e., the "no centrifugation" or "NC" group), the total detectable levels of the mRASSF1A marker were approximately 29.2-fold and 7.1-fold (patients #1 and #2, respectively) compared to the conventional method involving only the "supernatant" fraction after centrifugation. Such results are in excellent agreement with the fetal Y-Chr DNA detection results shown in Table 2 above.

[0101] For the mTERT marker, both patients (#3 and #4) were shown to be below the limit of detection (i.e., BLOD) in the "supernatant" fraction after centrifugation, indicating that the mTERT marker is virtually not detected by conventional cfDNA isolation and detection assays using centrifugation. In patient #3, the mTERT marker was also shown to be below the limit of detection (i.e., BLOD) in the "pellet" fraction after centrifugation, but when the mTERT marker was detected by an assay method that did not involve centrifugation (i.e., the "no centrifugation" or "NC" group), approximately 6 copies of the mTERT marker were detected, which was at least 6 times higher than the "supernatant" group. In patient #4, significantly more copies of the marker (i.e., 13.5) were detected in the "pellet" fraction after centrifugation, and even more copies (i.e., 16.5) were detected in the "NC" group. Thus, the data from both patients further indicated that when the assay was performed without centrifugation, the total detectable level of the mTERT marker was more than 6 times higher than the conventional method that essentially involved only the "supernatant" fraction after centrifugation.

[0102] HCC-associated DNA markers were detected in all pellet fractions. This data indicates that pre-centrifugation to remove cell pellets from urinary cfDNA isolation may result in loss of transrenal DNA of interest. Because different urine fractions from the same individual may contain different amounts of cellular debris, we did not compare DNA marker abundance between fractions from the same patient.

[0103] Discussion

[0104] As shown in Examples 1 and 2, transrenal fetal cfDNA is detectable in almost all samples or fractions of maternal urine samples, except for the post-wash cell pellet sample in Table 1 of Example 2. Even more surprisingly, as shown in Example 2, the "wash-off" fraction contains a significant amount of fetal cfDNA, which is at least 1 / 3 of the "supernatant" fraction of the Collection #1 sample, and even 1.26 times the "supernatant" fraction of the Collection #2 sample. These results indicate that the pre-wash pellet (Example 2) or pellet (Example 1) fractions, which are essentially pellets that are usually discarded after conventional centrifugation pretreatment of urine samples, contain a surprisingly significant portion of fetal cfDNA. This indicates that a portion of the transrenal circulating extracellular DNA molecules are associated with cells or cell debris in the collected urine sample, and these cells are pelleted together with the cell pellet after a short centrifugation at a typical pretreatment centrifugation speed of 1,000 rpm for 10 minutes or faster.

[0105] As further shown in Table 2, the Y-Chr copies recovered in the NC samples are about 4-5 times higher than those in the centrifugation-pretreated samples of all fractions combined, so the NC approach surprisingly seems to be much more efficient at recovering fetal cfDNA, an unambiguous transrenal DNA marker, from maternal urine samples than the centrifugation-wash-clearance approach. For example, in collection #1, the difference between the NC sample (i.e., 49.58) and the sum of the centrifugation-pretreated samples (i.e., 7.99 + 2.78 = 10.77) is about 4.6-fold. In collection #2, the difference between the NC sample (i.e., 17.62) and the sum of the centrifugation-pretreated samples (i.e., 1.51 + 1.91 = 3.42) is about 5.2-fold. This is presumably due to DNA loss during each pretreatment step, including the washing-clearance of the pellet, and the latter isolation step. Therefore, to recover transrenal cfDNA from urine samples, a method that does not involve centrifugation as a pretreatment and directly isolates DNA from the collected urine sample is considered desirable. Notably, exogenous spiked cfDNA, such as dsDNA SPKN 131bp DNA fragment, has little or no significant association with the cell pellet and remains in the supernatant, which can be used as a fractionation control of the supernatant from the cell pellet. This indicates that not all types of cfDNA are associated to the same extent with the cell debris pellet resulting from pretreatment centrifugation. Example 3 provides another transrenal cfDNA, HCC-associated DNA marker, which was also detected in the pellet fraction after pretreatment centrifugation. Considering the physicochemical properties similar to cfDNA, centrifugation as a pretreatment step may also pull down a significant portion of HCC-derived DNA markers or other endogenous cell-free nucleic acids (cfNAs), such as miRNA and cfRNA, in addition to cfDNA, together with the pellet obtained thereby.

[0106] conclusion

[0107] When circulating extracellular fetal DNA and liver-derived DNA, including hepatocellular carcinoma-derived DNA and HBV DNA, were examined in urine, two surprising and important observations were obtained. First, a significant amount of circulating-derived cell-free DNA (cfDNA) or transrenal DNA in urine is associated with the cell pellet generated by centrifugation. As a result, pretreatment using centrifugation to obtain supernatant for urinary cfDNA isolation may result in a substantial loss of cfDNA of interest, such as fetal DNA, HCC DNA, colorectal cancer DNA, and HBV DNA, as well as other transrenal DNA, if DNA isolation is performed only from the supernatant. Second, circulating-derived cfDNA or transrenal DNA in urine is low molecular weight (LMW) DNA with a relatively small size of less than 1 kb, whereas genomic DNA obtained in cells is high molecular weight (HMW) DNA with a much larger size. To isolate cfDNA in body fluids such as urine without reprocessing, it is necessary to remove HMW DNA, which is the majority of background DNA, after isolating total urinary DNA, thereby increasing the concentration of the DNA of interest and improving detection sensitivity. Selective enrichment of circulating-derived cfDNA in urine or other bodily fluids by size fractionation and removal of HMW DNA to obtain LMW DNA can be achieved by a variety of methods including, but not limited to: use of chromatography or electrophoresis such as chromatography on agarose or polyacrylamide gels, ion-pair reversed-phase high performance liquid chromatography, autogenous coating, capillary electrophoresis in low viscosity or other polymer matrices, selective extraction on microfabricated electrophoresis devices, microchip electrophoresis, adsorbent membrane chromatography; density gradient centrifugation, and methods utilizing nanotechnology means such as microfabricated entropy trap arrays, carboxylated beads, etc.

[0108] In this way, the LMW DNA fraction obtained by isolating total DNA and then removing HMW DNA without using centrifugation or other means of recovering cellular debris not only allows the subsequent determination of the genetic traits of the fetus in pregnancies at risk of inherited diseases, sex, or cancer genetics for early cancer detection, cancer screening, and detection of viral genetics for disease management or disease control.

[0109] Determination of such genetic characteristics of interest can be carried out by methods such as polymerase chain reaction (PCR) technology, probe hybridization, next-generation sequencing, and nucleic acid arrays (such as DNA chips).

[0110] References 1. Chan, AK, RW Chiu, and YD Lo, Cell-free nucleic acids in plasma, serum and urine: a new tool in molecular diagnosis. Annals of clinical biochemistry, 2003. 40(2): p. 122-130. 2. Tsui, NB, et al., High resolution size analysis of fetal DNA in the urine of pregnant women by paired-end massively parallel sequencing. PloS one, 2012. 7(10): p. e48319. 3. Botezatu, I., et al., Genetic analysis of DNA excreted in urine: a new approach for detecting specific genomic DNA sequences from cells dying in an organism. Clinical chemistry, 2000. 46(8): p. 1078-1084. 4. Al-Yatama, M.K., et al., Detection of Y chromosome-specific DNA in the plasma and urine of pregnant women using nested polymerase chain reaction. Prenatal diagnosis, 2001. 21(5): p. 399-402. 5. Majer, S., et al., Maternal urine for prenatal diagnosis-an analysis of cell-free fetal DNA in maternal urine and plasma in the third trimester. Prenatal diagnosis, 2007. 27(13): p. 1219-1223. 6. Li, Y., et al., Inability to detect cell free fetal DNA in the urine of normal pregnant women nor in those affected by preeclampsia associated HELLP syndrome. Journal of the society for gynecologic investigation, 2003. 10(8): p. 503-508. 7. Illanes, S., et al., Detection of cell-free fetal DNA in maternal urine. Prenatal Diagnosis, 2006. 26(13): p. 1216-1218. 8. Koide, K., et al., Fragmentation of cell-free fetal DNA in plasma and urine of pregnant women. Prenatal diagnosis, 2005. 25(7): p. 604-607. 9. Shekhtman, E.M., et al., Optimization of transrenal DNA analysis: detection of fetal DNA in maternal urine. Clinical chemistry, 2009. 55(4): p. 723-729. 10. Lin, S.Y., et al., A New Method for Improving Extraction Efficiency and Purity of Urine and Plasma Cell-Free DNA. Diagnostics, 2021. 11(4): p. 650-659. 11. Lin, S.Y., et al., Detection of Hepatitis B Virus-Host Junction Sequences in Urine of Infected Patients. Hepatology communication, 2021. 5(10): p. 1649-1659. 12. Jain, S., et al., Characterization of the hepatitis B virus DNA detected in urine of chronic hepatitis B patients. BMC gastroenterology, 2018. 18(1): p. 40. 13. Lin, S.Y., et al., Detection of CTNNB1 Hotspot Mutations in Cell-Free DNA from the Urine of Hepatocellular Carcinoma Patients. Diagnostics, 2021. 11(8): p. 1475. 14. Hann, H.W., et al., Detection of urine DNA markers for monitoring recurrent hepatocellular carcinoma. Hepatoma research, 2017. 3: p. 105-111. 15. Zhang, A., et al. Urine as an Alternative to Blood for Cancer Liquid Biopsy and Precision Medicine. in 2018 IEEE International Conference on Bioinformatics and Biomedicine (BIBM). 2018. IEEE. 16. Kim, A.K., et al., Urine DNA biomarkers for hepatocellular carcinoma screening. British journal of cancer, 2022. 126: p. 1432-1438. 17. Su, Y.H., et al., Human urine contains small, 150 to 250 nucleotide-sized, soluble DNA derived from the circulation and may be useful in the detection of colorectal cancer. Journal of nolecular diagnostics, 2004. 6(2): p. 101-107. 18. Su, Y.H., et al., Detection of K-ras mutation in urine of patients with colorectal cancer. Cancer biomarkers, 2005. 1: p. 177-182. 19. Song, B.P., et al., Detection of hypermethylated vimentin in urine of patients with colorectal cancer. Journal of molecular diagnostics, 2012. 14(2): p. 112-119.

Claims

**Claim 1** A method for characterizing a target cell-free nucleic acid molecule present in a biological sample, the method comprising: isolating total cell-free nucleic acid from the biological sample without performing a pretreatment to remove cell debris from the biological sample; and characterizing the target cell-free nucleic acid molecule from the isolated total cell-free nucleic acid; wherein the method is capable of detecting at least twice as many copies of the target cell-free nucleic acid molecule in the biological sample as compared to when cell debris is removed by pretreatment in the isolating step. A method. **Claim 2** The method according to claim 1, wherein the biological sample comprises a urine sample, a serum sample, a plasma sample, a saliva sample, a sweat sample, a lymph fluid sample, or any combination thereof. **Claim 3** The method according to claim 2, wherein the biological sample is a urine sample and the target cell-free nucleic acid molecule comprises at least one of a renal nucleic acid molecule or an apoptosis-derived nucleic acid molecule. **Claim 4** The method according to claim 1, wherein the target cell-free nucleic acid molecule is a target cell-free DNA molecule, the step of isolating total cell-free nucleic acid from the biological sample comprises isolating total cell-free DNA from the urine sample, and the step of characterizing the target cell-free nucleic acid molecule from the isolated total cell-free nucleic acid comprises characterizing the target cell-free DNA molecule from the isolated total cell-free DNA. A method. **Claim 5** The method according to claim 4, wherein the target cell-free DNA molecule has a length shorter than about 1 kb, and the method further comprises, after the step of isolating total cell-free DNA from the urine sample and before the step of characterizing the target cell-free DNA molecule from the isolated total cell-free DNA, obtaining low molecular weight DNA from the isolated total cell-free DNA, wherein the step of characterizing the target cell-free DNA molecule from the isolated total cell-free DNA comprises characterizing the target cell-free DNA molecule from the low molecular weight DNA. A method. **Claim 6** The method according to claim 5, wherein the step of obtaining low molecular weight DNA from the isolated total cell-free DNA is performed by a size fractionation approach selected from a carboxylated magnetic bead-based approach, an agarose gel-based chromatography approach, or a polyacrylamide gel-based chromatography approach. **Claim 7** The method according to claim 5, wherein the step of characterizing the target cell-free DNA molecule from the low molecular weight DNA is performed by at least one of a polymerase chain reaction (PCR) assay, a sequencing assay, or a hybridization assay.

8. The method according to claim 5, wherein the target cell-free DNA molecule is a cancer-related DNA marker selected from mutated K-ras, methylated RASSF1A (mRASSF1A), or mutated TERT (mTERT).

9. The method according to claim 5, wherein the urine sample is collected from a pregnant woman, and the target cell-free DNA molecule is a fetal DNA marker related to gender, autosomal traits, or genetic diseases.

10. The method according to claim 9, wherein the fetal DNA marker includes a Y chromosome (Y-Chr) marker.

11. The method according to claim 5, wherein the target cell-free DNA molecule is a DNA marker of a microorganism, and the microorganism is a virus, a bacterium, or a fungus.

12. The method according to claim 11, wherein the target cell-free DNA molecule is a DNA marker of hepatitis B virus (HBV).

13. In the method according to claim 1, the target cell-free nucleic acid molecule is target cell-free RNA, the step of isolating total cell-free nucleic acid from the biological sample includes the step of isolating total cell-free RNA from the urine sample, and the step of characterizing the target cell-free nucleic acid molecule from the isolated total cell-free nucleic acid includes the step of characterizing the target cell-free RNA molecule from the isolated total cell-free RNA. Method.

14. The method according to claim 13, wherein the target cell-free RNA molecule includes microRNA.

15. The method according to claim 1, wherein the step of isolating total cell-free nucleic acid from the biological sample is performed using carrier RNA.

16. The method according to claim 1, wherein the method can detect at least 6-fold more copies of the target cell-free nucleic acid molecule in the biological sample compared to the case where cell debris is removed by pretreatment in the isolation step.

17. A method for characterizing a target cell-free nucleic acid molecule in a biological sample, the method comprising: a step of pretreating the biological sample, A step of centrifuging the biological sample to obtain a supernatant fraction and a pellet fraction; a step of washing the pellet fraction with a washing solution to obtain a washing-removed fraction; and a step of combining the supernatant fraction and the washing-removed fraction to obtain a composite fraction, A step of pretreating; A step of isolating total cell-free nucleic acid from the composite fraction; and A step of characterizing the target cell-free nucleic acid molecule from the isolated total cell-free nucleic acid comprising The method can detect at least 1.25 times as many copies of the target cell-free nucleic acid molecule from the biological sample as compared to the case where the total cell-free nucleic acid is isolated only from the supernatant fraction of the pretreated biological sample. Method.

18. The method according to claim 17, wherein the washing solution has a pH of about 2.0 to 4.

0.

19. In the method according to claim 17, the cleaning liquid contains a salt having a concentration of about 0.15 to 3 M, and the salt is NaCl, KC1, MgCl ２ , LiCl, sodium citrate, or any combination thereof.

20. The method according to claim 17, wherein the method can detect at least 2 times as many copies of the target cell-free nucleic acid molecule from the biological sample as compared to the case where the total cell-free nucleic acid is isolated only from the supernatant fraction of the pretreated biological sample.

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