Protocol for miRNA isolation from biofluids

Analytical Biochemistry 499 (2016) 78e84

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Protocol for miRNA isolation from biofluids Evgeny A. Lekchnov a, *, Ivan A. Zaporozhchenko a, b, Evgeny S. Morozkin a, b, Olga E. Bryzgunova a, Valentin V. Vlassov a, Pavel P. Laktionov a, b a b

Institute of Chemical Biology and Fundamental Medicine of SB RAS, Novosibirsk 630090, Russia Novosibirsk Research Institute of Circulation Pathology of Academician E.N. Meshalkin, Novosibirsk 630055, Russia

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 December 2015 Received in revised form 27 January 2016 Accepted 29 January 2016 Available online 11 February 2016

MicroRNAs (miRNAs) have been identified as promising biomarkers in cancer and other diseases. Packaging of miRNAs into vesicles and complexes with proteins ensures their stability in biological fluids but also complicates their isolation. Conventional protocols used to isolate cell-free RNA are generally successful in overcoming these difficulties; however, they are costly, labor-intensive, or heavily reliant on the use of hazardous chemicals. Here we describe a protocol that is suitable for isolating miRNAs from biofluids, including blood plasma and urine. The protocol is based on precipitation of proteins, denaturation of miRNA-containing complexes with octanoic acid and guanidine isothiocyanate, and subsequent purification of miRNA on spin columns. The efficacy of miRNA extraction by phenolechloroform extraction, miRCURY RNA isolation kitdbiofluids (Exiqon), and the proposed protocol was compared by quantitative reverse-transcription PCR of miR-16 and miR-126. The proposed protocol was slightly more effective for isolating miRNA from plasma and significantly superior to the other two methods for miRNA isolation from urine. Spectrophotometry and SDS-PAGE data suggest that the disparity in performance between miRCURY Biofluids and the proposed protocol can be attributed to differences in precipitation mechanisms, as confirmed by the retention of different proteins in the supernatant. © 2016 Elsevier Inc. All rights reserved.

Keywords: Cell-free miRNA miRNA isolation Plasma Urine

Introduction MiRNAs have been shown to function not only inside cells as post-transcriptional regulators of gene expression, but also as messengers of intracellular communication [1]. Encapsulated in exosomes and other microvesicles, or bound in complexes with proteins, miRNAs are secreted into the extracellular space, where they are transported to near and distant cells, enter biological fluids, and travel throughout the entire organism [2]. The presence and high stability of miRNAs in biological fluids, reliable proof of the presence of tissue- or tumor-specific miRNA in the total pool of cell-free miRNA, and the ease of quantitative miRNA detection promote their use as perspective diagnostic targets [3]. Indeed, detection of certain cell-free miRNAs has been shown to be an effective tool for diagnosing tumor development, therapy monitoring, relapses, prognosis, and theranostics [4e7].

* Corresponding author. Current address: Laboratory of Molecular Medicine, ICBFM SB RAS, Novosibirsk 630090, Russia. E-mail address: [email protected] (E.A. Lekchnov). http://dx.doi.org/10.1016/j.ab.2016.01.025 0003-2697/© 2016 Elsevier Inc. All rights reserved.

Cell-free miRNAs are stable in a variety of biological fluids including blood, lymph, cerebrospinal liquid, bronchoalveolar lavage, urine, saliva, and tears [8]. Their stability is attributed to the engagement of miRNA in complexes with proteins [9], lipoproteins [10], and membrane-coated vesicles such as microparticles, exosomes, and apoptotic bodies [11]. Analysis of cell-free miRNAs requires their extraction from biological fluids; however, the stability of complexes containing miRNA complicates their isolation and subsequent analysis. Not all protocols conventionally used for isolation of cell-free RNA and DNA from blood are suitable for miRNAs [7,12e14]. Acid phenolechloroform extraction has been considered to be a “gold standard” of RNA isolation. A significant number of improvements and modifications adjusting the protocol to specific applications have been proposed, but the core principle has remained unchanged [15]. It has proved to be efficient, but also labor-intensive, and relies heavily on the use of hazardous chemicals, such as phenol, which makes it inconvenient for common use. More recently, a new high-performance approach to RNA isolation from biofluids has been reported (miRCURY, Exiqon) [7]. A significant effort of recent cell-free miRNA studies is directed at the investigation of miRNA levels in blood, and other biofluids

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(such as urine) attract increasing attention, especially in regard to the detection of bladder and prostate tumors [6,16,17]. Therefore, the lack of simple, standardized protocols for miRNA extraction and analysis hinder the development of miRNA-based therapeutics and diagnostics and their introduction into routine clinical laboratory practice. An efficient, reliable, and standardized protocol for cell-free miRNA isolation from different biological fluids that will make the procedure less time-consuming, less cumbersome, and easily adaptable to automated systems and clinical needs is demanded by researchers and clinicians alike. Here, we describe a fast and simple protocol for miRNA extraction based on precipitation of proteins and denaturation of miRNA-containing complexes with octanoic acid (OcA) and guanidine isothiocyanate (Gu), with subsequent purification of miRNA using spin columns.

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dried at 10,000 g for 1 min. The columns were discarded; the flowthrough contained purified miRNA. The miRNAs were precipitated with glycogen as described below. Gu/OcA protocol for miRNA isolation from urine A sample of 400 ml of urine was mixed with 10 ml of 2-mercaptoethanol, 200 ml of denaturing buffer comprised of 0.3e1.35 M Gu, 400 ml of precipitation buffer comprising of 0.8 M sodium acetate pH 4.0 and 0.5e2.5% OcA [optimal conditions 1.35 M Gu, 1.5% OcA (pH 4.0); see Results and discussion] listed in Table S2 of the Supplementary Materials. Subsequent isolation steps were performed according to the previously described procedure for blood plasma.

Materials and methods

Acid phenolechloroform RNA isolation

Samples of biological fluids

RNA was isolated from 150 ml of blood plasma or 400 ml of clarified urine. Acid phenolechloroform extraction was performed as described in Chomczynski and Sacchi [15]. The 150 ml of plasma was mixed with 4.5 ml of 2-mercaptoethanol and 300 ml of denaturing buffer (6.75 М Gu) in a 2-ml Eppendorf propylene tube. The mixture was vortexed for 5 s. Then 45 ml of 2 M sodium acetate (pH 4.0), 450 ml of water-saturated phenol, and 90 ml of chloroform/ isoamyl alcohol (49:1) were added. The mixture was vortexed for 5 s, incubated for 15 min on ice, and centrifuged at 10,000 g for 20 min at 4  C. After phase separation, the water phase was collected, mixed with an equal volume of ethanol, and purified using silica columns (BioSilica Ltd., Novosibirsk, Russia). Briefly, the sample was applied to a silica spin column under a vacuum manifold or alternatively in a benchtop centrifuge, no more than 500 ml at a time. The flowthrough was discarded. The column was washed twice with 300 ml of washing buffer (1 M Gu, 2.5 mM Tris-acetate, 50% ethanol, 25% chloroform, 1% 2-mercaptoethanol). After the second washing, the column was dried by centrifugation at 10,000 g for 1 min. The flowthrough was discarded, and the column was washed twice with 300 ml of washing buffer (10 mM TriseHCl (pH 7.5), 0.1 M NaCl, 75% ethanol). After the second washing, the column was dried by centrifugation at 10,000 g for 1 min. The flowthrough was discarded. RNA was eluted from the column with 120 ml of BioSilica RNA elution solution with 10 mM EDTA (рН 9.5) as described above.

Blood and urine samples from 10 healthy individuals were obtained from the Novosibirsk Research Institute of Circulation Pathology of Academician E.N. Meshalkin (Novosibirsk, Russia) and the Center of New Medical Technologies of ICBFM SB RAS (Novosibirsk, Russia) after approval of the study by the ethics committees of both organizations. Written informed consent was provided by all participants. Venous blood was collected in EDTA spray-coated vacutainers (BD, Cat. No. 368589) and fractionated into plasma and blood cells within 4 h of blood sampling. Blood was centrifuged at 290 g for 20 min, transferred into new tubes, and centrifuged a second time at 1200 g for 20 min. Urine was collected from participants and immediately clarified by two serial centrifugations at 400 g and 17,000 g, both at 20  C for 20 min. Supernatants of plasma and urine samples from the 10 donors were stored frozen in aliquots at 20  C. Before isolation of miRNA, the blood plasma or urine samples were thawed, mixed gently, and centrifuged at 3000 g for 5 min. Gu/OcA protocol for miRNA isolation from plasma A sample of 150 ml of plasma was mixed with 4.5 ml of 2mercaptoethanol, 100 ml of denaturing buffer comprising 0.3e1.35 M guanidine thiocyanate (Gu), 150 ml of precipitation buffer comprising 2.0 M sodium acetate pH 4.0e6.0 and 0.5e2.5% OcA (optimal conditions 0.6 M Gu, 0.5% OcA, pH 4.0; see Results and discussion) listed in Table S1 in the Supplementary Material. The mixture was vortexed for 5 s and incubated for 3 min at room temperature. Before introduction into silica columns, the samples were centrifuged in a benchtop centrifuge at maximum speed (approx. 10,000 g) for 3 min. Then the supernatant was mixed thoroughly with an equal volume of 95% ethanol and introduced into a silica spin column (e.g., BioSilica Ltd., Novosibirsk, Russia) under a vacuum manifold or alternatively in a benchtop centrifuge, no more than 500 ml at a time. The flowthrough was discarded. The columns were washed twice with 300 ml of washing buffer containing 5 M Gu, 10 mM Tris-acetate (pH 6.5), 50% ethanol, and 1% 2-mercaptoethanol. After the second washing, the columns were dried by centrifugation at 10,000 g for 1 min. The flowthrough was discarded. The columns were washed twice with 300 ml of washing buffer (10 mM TriseHCl (pH 7.5), 0.1 M NaCl, 75% ethanol). After the second washing, the columns were dried by centrifugation at 10,000 g for 1 min. The flowthrough was discarded. To elute the RNA from the column, 120 ml of BioSilica RNA elution solution with 10 mM EDTA (рН 9.5) was applied to the center of the filter and centrifuged at 400 g for 15 min and was then

Exiqon miRCURY biofluids kit for RNA isolation Isolation from blood plasma and urine was performed using the Exiqon miRCURY biofluids kit (#300112) according to the manufacturer's protocols. An additional step of RNA precipitation as described below was performed to ensure miRNA purity. RNA precipitation To precipitate the miRNA, 2 ml of glycogen (20 mg/ml) and 12 ml of 3 M sodium acetate (pH 7.5) were added to 120 ml of RNA eluate and mixed by vortexing. Then 132 ml of isopropanol was added and was mixed by inversion and then by vortexing. The mixture was incubated for 30 min at 20  С and then centrifuged at 12,000 g for 10 min at 4  С. The supernatant was discarded. The pellet was washed with 75% ethanol at 7500 g for 5 min at 4  С. The supernatant was discarded. Then the pellet was washed with 96% ethanol at 7500 g for 5 min at 4  С. The supernatant was discarded, and the pellet was air-dried and dissolved in 15 ml of RNAse-free water.

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RNA analyses The size distribution of RNA isolated from blood and urine using the Gu/OcA protocol was analyzed using capillary electrophoresis (Agilent 2100 Bioanalyzer, Agilent Technologies, USA). RNA samples (2 ml) were analyzed with Agilent small RNA analysis kits according to the manufacturer's protocol. Electropherograms were analyzed using Agilent 2100 Expert B.02.07 software. Reverse transcription and quantitative RT-PCR Reverse transcription (RT) on miRNA templates was performed as described by Chen et al. [18]. Primers and probes for reverse transcription and TaqMan qPCR (Supplementary Materials, Table S2) were synthesized in the Laboratory of Medicinal Chemistry (ICBFM SB RAS, Novosibirsk). Each RT reaction was performed in a total volume of 20 ml and contained 5 ml of RNA, 50 nM each of miRNA-specific primers, 1 unit of RiboLock RNAse inhibitor (Fermentas, Vilnius, Lithuania), 100 units of MMLV reverse transcriptase (Fermentas, Vilnius, Lithuania), 4 ml of 5  MMLV reaction buffer (250 mM TriseHCl (pH 8.3 at 25  C), 250 mM KCl, 20 mM MgCl2, 50 mM DTT), and 250 mM of each dNTP. The reaction conditions were as follows: 16  C for 30 min, 42  C for 30 min, and 70  C for 10 min. Samples without RNA templates and preparations of genomic DNA were used as negative controls. Real-time PCR was carried out on the iCycler iQ5 real-time PCR detection system (Bio-Rad, USA). All reactions were carried out in duplicate in a total volume of 30 ml. Each reaction contained 5 ml of RT product, 1.25 unit of Taq DNA polymerase (BiolabMix, Russia), 3 ml of 10  PCR buffer (750 mM TriseHCl (pH 8.8 at 25  C), 200 mM (NH4)2SO4, 0.1% (v/v) Tween 20), 4 mM MgCl2, 250 mM of each dNTP, 600 nM forward primer, 800 nM universal reverse primer, and 300 nM specific TaqMan probe (Supplementary Materials, Table 2). After an initial denaturation at 95  C for 3 min, the reactions were run for 50 cycles at 95  C for 15 s and 60  C for 45 s. Laemmli SDS PAGE and Western blot The samples were separated on 10e20% polyacrylamide gels (PAA ratio 30:1) as described elsewhere by SDS PAGE using Laemmli buffers and 4.5% concentrating gels [19]. Before application to the gel, the samples were incubated at 95  C for 5 min in buffer (62.5 mM TriseHCl (pH 6.8), 10% glycerol, 0.5% SDS, 2.5% 2-mercaptoethanol) containing bromophenol blue and then centrifuged for 5 min at 17,000 g. Proteins were transferred from PAA gels to nitrocellulose membranes in the Western blot buffer (217 mM Tris-glycine (pH 8.6), 9.5% ethanol), and the membrane was stained with colloidal silver as previously described [20]. Results and discussion It was shown more than 50 years ago that at low (0.04 M) concentration octanoic acid (and several other fatty acids) forms insoluble complexes with the majority of blood plasma proteins including a- and b-globulins, and at higher concentrations, with albumins and globulins [21]. Due to this, OcA was successfully used for purification of immunoglobulins from blood plasma [22,23]. Since the chaotropic effects of Gu alone fail to destroy complexes of miRNA with proteins and other biopolymers completely [24], the addition of compounds preventing hydrophobic interactions is required to release miRNA from complexes [25]. Because of the OcA affinity to hydrophobic moieties (ability to form/destroy hydrophobic interactions), it is capable of efficient binding/destruction of biological membranes and lipoprotein complexes. Thus, we propose to use the combination of two denaturating/precipitating

agentsdGu and OcA. The precipitation of proteins or destruction of circulating miRNA complexes and membrane-enveloped microparticles in the presence of both OcA and Gu is difficult to predict. Therefore, a set of conditions were tested. To estimate the efficacy of the protocol, different concentrations of Gu and OcA were added to 200 ml of blood plasma or 400 ml of urine. The residual amount of protein in the supernatant was determined using a Genesys 10 UV spectrophotometer (Thermo Scientific, USA) as the absorbance at 260 and 280 nm. Before the measurements, samples were diluted 10 times with 0.02 M Tris/HCl and 0.15 M NaCl pH 7.5 (TBS). The dependence of the efficacy of protein precipitation on pH and the proportions of both chemical agents, as well as miRNA yield at different pH and concentrations of Gu and OcA, were tested. The concentrations of miR-16 and miR-126 in supernatants were measured by quantitative RT-PCR. The Ct values of the miRNAs and the absorbance values were first considered to identify the optimal conditions for miRNA isolation from plasma and urine. The data presented in Fig. 1 clearly demonstrate that isolation of miRNA from plasma is most efficient at pH 4.0 and in the presence of 0.6 M Gu and 0.5% OcA. When 0.3 M Gu is used at the same pH and OcA concentration, the supernatant contains less protein, with a significant portion of miRNA supposedly co-precipitating with the pellet. In contrast, at higher Gu concentrations, the proteins were precipitated less effectively by OcA and thus the miRNA yield was reduced. The efficacy of miRNA extraction was close to optimal at 0.9 M Gu, 0.5% OcA, and pH 5.0; however, the reproducibility was low and the reagent consumption was considerably higher. It is worth noting that in the presence of 0.6 M Gu and 0.5% OcA at pH 4.0, the residual amount of protein detected in blood plasma supernatants was minimal (Table S1 in the Supplementary Material). In contrast to isolation from plasma, several sets of conditions for miRNA isolation from urine resulted in high yields. The optimal conditions were determined to be 1.35 M Gu and 1.5% OcA at pH 4.0 (Fig. 2). Under these conditions, the greatest amount of urine protein was removed from the supernatant (Table S2 in the Supplementary Material). These experimental data demonstrate that a combination of OcA and Gu can successfully release miRNA from complexes present in the blood and urine and provide efficient precipitation of excess biopolymers. It has previously been shown that the ability of OcA to precipitate proteins depends on the sample dilution, pH values, OcA concentration, and temperature [26]. The major portion of the nonimmunoglobulin protein fraction is precipitated by octanoic acid at pH 4.2 and 4.5 [21,22]. Increasing the pH to 5.1 results in a significant decrease in precipitation. Albumin and alpha and beta globulins precipitate completely at 20e30 ml/ml (2e3%) OcA concentration and 22  C [26]. It was shown that van der Waals forces are involved in the interaction of proteins with OcA, and tyrosine moieties in proteins play a crucial role in this process [27,28]. To assess the size and quality of miRNA obtained with the OcA protocol, preparations of RNA isolated from blood plasma and urine were analyzed using an Agilent 2100 Bioanalyzer (small RNA analysis kits). The Agilent small RNA kit enables the detection of small RNA in the interval 6e150 nucleotides, including the miRNA region (18e25 nucleotides). Bioanalyzer electropherograms showed the presence of primarily short RNA molecules (10e50 nt) in samples derived from blood plasma and urine using the Gu/OcA protocol (Fig. 3). Thus, the experimental results demonstrate that the Gu/OcA protocol allows the efficient isolation of microRNA from biofluids. Nevertheless, the efficacy of miRNA isolation must be investigated in a comparative study. Thus, we evaluated the efficacy of miRNA extraction from blood and urine by phenolechloroform

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Fig.1. Dependence of miRNA yield on Gu concentration and pH for 0.5% OcA. Plasma samples of 10 healthy individuals were pooled and used for all isolations. The graph shows the mean Ct and standard deviation (Ct SD). The asterisk (*) indicates optimal conditions for miRNA isolation.

Fig.2. MiRNA isolation efficacy depending on Gu concentration and pH for 1.5% OcA. Urine samples of 10 healthy individuals were pooled and used for all isolations. The graph shows the mean Ct and standard deviation (Ct SD). The asterisk (*) indicates optimal conditions for miRNA isolation.

extraction [15], miRCURY RNA isolation kitdbiofluids (Exiqon) [29,30], and the Gu/OcA protocol. MiRNA was isolated from 10 samples of blood plasma and 10 samples of urine obtained from different healthy donors by the protocols described in Materials and methods. All manipulations with the miRCURY RNA isolation kitdbiofluids were performed according to the manufacturer's recommendations. In each case, the isolated microRNAs were co-precipitated with glycogen. To estimate the performance of the protocols, we measured the expression levels of two miRNAs (miR-16 and miR-126) as some of the most abundant miRNAs in blood plasma. Threshold cycle (Ct) values of miR-16 and miR-126 were compared in samples isolated by different protocols from biofluids obtained from the same individuals. The Gu/OcA protocol outperformed all other methods in miRNA isolation from human blood plasma. The Gu/OcA protocol was 7.21-fold more efficient than acid phenolechloroform extraction and 1.65-fold more efficient than miRCURY Biofluids at isolating miR-16. The isolation efficacy of miR-126 by the Gu/OcA method was also superior to acid

phenolechloroform extraction and miRCURY Biofluids (1.43- and 2.85-fold, respectively). The Gu/OcA protocol has also proven to be superior to other isolation methods at miRNA isolation from urine samples. It was 2.29 times more effective than acid phenolechloroform extraction as measured by miR-16 yields. In the case of miR-126, acid phenolechloroform extraction underperformed the octanoic acidbased method more than 1.47-fold. The Gu/OcA protocol also significantly surpassed the miRCURY biofluids kit in miRNA isolation from urine. MiR-16 yields obtained by Gu/OcA were 142-fold higher than after isolation using the miRCURY biofluids kit. Both Gu/OcA and acid phenolechloroform reliably isolated miR-126 from urine, while the presence of miR-126 could not be detected in RNA samples after isolation with miRCURY biofluids (Table 1). Since Gu/OcA protocol and miRCURY biofluids share a common fundamental approach to miRNA isolation (although the removal/ depletion of proteins is achieved by different chemical agents), we were interested to understand what caused such drastic differences in their performance. To compare the efficacy of protein removal by

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Fig.3. Bioanalyzer analysis of miRNA samples obtained from blood plasma (A) and urine (B). The electropherograms show the fluorescence intensity and size distribution in nucleotides (nt) of miRNA. The peak at 4 nt is an internal standard.

the miRCURY biofluids kit and the Gu/OcA protocol, we measured the absorbance values of supernatants produced by both methods at 260 and 280 nm and investigated the remaining proteins by SDS PAGE. Chemical agents used in the miRCURY biofluids kit remove proteins from blood with higher efficacy than the Gu/OcA protocol (A260/A280 0.45/0.16 versus 0.54/0.27). In contrast, Gu/OcA is more efficient in the elimination of biopolymers from urine (A260/ A280 for Gu/OcA is 0.88/0.73 versus 1.12/0.88). SDS PAGE identified one minor 52-kDa protein in the supernatant of blood plasma treated with the miRCURY biofluids kit (presumably antithrombin III, carboxypeptidase N catalytic chain, vitamin D-binding protein isoform 1 precursor, or other proteins [31,32]) and no less than 10 proteins, including albumin and IgG, in the supernatants after precipitation by Gu/OcA. These data demonstrate that more aggressive precipitation of the proteins by the miRCURY biofluids kit may lead to collateral co-precipitation of a portion of miRNAs. Obviously, the proteins remaining in the supernatant after Gu/OcA treatment do not interfere with miRNA isolation, at least in the presence of 0.6 M Gu, and are drastically different from the proteins retained in the sample after the precipitation step of miRCURY biofluids. The composition of the precipitation solution in the miRCURY biofluids kit is not available. However, our data suggest

that different precipitation approaches are used in the Gu/OcA method (octanoic acid) and miRCURY commercial kit (Fig. 4). Thus, our results show that Gu/OcA outperformed the other isolation methods and produced a high miRNA yield from plasma and urine (Table 1). In comparison to acid phenolechloroform extraction, miRCURY biofluids produced a higher efficiency of miR-

Table 1 Comparison of the efficacy of miRNA isolation from blood plasma and urine by different protocols. Gu/OcA

Blood plasma Urine a

Not detected.

miR-16 miR-126 miR-16 miR-126

miRCURY biofluids kit

Acid phenol echloroform extraction

Ct mean

Ct SD

Ct mean

Ct SD

Ct mean

Ct SD

21.92 30.66 29.8 34.16

1.66 0.64 1.93 1.94

22.64 32.17 36.95 NDa

1.48 1.43 1.03 ND

22.77 31.18 31 34.72

1.53 0.27 1.03 1.14

Fig.4. SDS-PAGE of human serum precipitated by miRCURY biofluids protein precipitation solution and Gu/OcA. 1, blood plasma; 2, supernatant of blood plasma treated with Gu/OcA; 3, supernatant of blood plasma treated with the miRCURY biofluids kit.

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16 isolation from plasma but yielded significantly less miRNA when urine was used for isolation. To date, acid phenolechloroform extraction is the most commonly used method for miRNA isolation from different biological sources. Along with its advantages, this method has some significant limitations. The procedure is time-consuming (40e60 min) and requires high-quality reagents (phenol), and the results depend significantly on the skill of the investigator. In addition, phenol is a highly toxic reagent, which requires additional equipment and waste management systems. This creates significant difficulties for its routine use [33]. Moret et al. compared four different protocols for RNA extraction using TRIzol-LS, a mirVana PARIS kit, a miRNeasy serum/ plasma kit, and TRIzol-LS with a mirVana kit from fresh and frozen plasma samples. They demonstrated that the column-based methods were more effective than TRIzol due to the presence of organic contaminants in RNA preparations obtained by TRIzol isolation [34]. Moreover, it was shown that miRNAs with low GC content and secondary structure (e.g., miR-141 and miR-21) are isolated less efficiently, and miRNA isolation from biological fluids is also lacking [35]. The miRCURY biofluids kit (Exiqon, Denmark) has been developed relatively recently as an alternative to current phenol-based commercial methods. Its advantages are the absence of phenol and the lack of a laborious phase separation step, which reduces the time required for isolation and simplifies its use [33]. According to published data, miRCURY biofluids surpasses other commercial kits for miRNA isolation from plasma, including Trizol LS, and results in high recovery of miRNA [29,30]. miRCURY biofluids produced the highest miRNA yield from both exosomes and cultured cells. This indicates high lysing capabilities, effective miRNA adsorption on spin columns, and high elution efficiency [30]. Cheng et al. compared six commercial RNA isolation kits including the miRCURY biofluids kit using exosomes obtained from urine [36]. The authors demonstrated that the miRCURY biofluids kit was less effective than the miRNeasy kit with RNeasy MinElute columns (with miRNA enrichment), the miRNeasy kit (without miRNA enrichment), and the Urine Exosome RNA isolation kit (Norgen, Biotek). However, the miRCURY biofluids kit isolation efficiency was comparable to that of mirVana PARIS (Ambion) and Trizol LS reagent (Life Technologies) with mirVana (Ambion). Comparison between published reports and the obtained data leads to the conclusion that miRCURY biofluids is more effective at miRNA extraction from blood plasma than from urine. Conclusions The Gu/OcA method enables fast and efficient isolation of miRNA from plasma and especially from urine. The resulting preparations of RNA are highly enriched in miRNA that is ready for quantitative RT-PCR analysis. The method can be performed at benchtop and easily adapted to various biological fluids. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ab.2016.01.025. References [1] M. Simons, G. Raposo, Exosomes e vesicular carriers for intercellular communication, Curr. Opin. Cell Biol. 4 (2009) 575e581, http://dx.doi.org/ 10.1016/j.ceb.2009.03.007. [2] J. Lin, J. Li, B. Huang, J. Liu, X. Chen, X.M. Chen, Y.M. Xu, L.F. Huang, X.Z. Wang, Exosomes: novel biomarkers for clinical diagnosis, Sci World J. 2015 (2015) e657086, http://dx.doi.org/10.1155/2015/657086.

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