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• W76546127 abstract "Circulating miRNAs are intensively evaluated as promising blood-based biomarkers. This growing interest in developing assays for circulating miRNAs necessitates careful consideration of the effects of preanalytical and analytical parameters on the isolation, stability, and quantification of circulating miRNAs. By using quantitative stem-loop RT-PCR, we compared the relative efficiencies of four miRNA isolation systems and different storage conditions. The effect of the data normalization procedure on the quantification of circulating miRNA levels in plasma from 30 healthy individuals and 30 patients with non–small cell lung carcinoma was estimated by measuring endogenous hsa-miR-21 and hsa-miR-16 and exogenous cel-miR-39 that was spiked in all samples at the same concentration. Silica column–based RNA extraction methods are more effective and reliable with respect to TRIzol LS. Endogenous circulating miRNA levels are unstable when plasma is stored at 4°C, and samples should be kept at −70°C, where the extracted miRNAs remain stable for up to 1 year. When normalization is based on combined endogenous and exogenous control miRNAs, differences in miRNA recovery and differences in cDNA synthesis between samples are compensated. Using this normalization procedure and hsa-miR-21 as a biomarker, we could clearly discriminate healthy individuals from patients with cancer. Experimental handling and the use of exogenous and endogenous controls for normalization are critical for the reliable quantification of circulating miRNA levels in plasma. Circulating miRNAs are intensively evaluated as promising blood-based biomarkers. This growing interest in developing assays for circulating miRNAs necessitates careful consideration of the effects of preanalytical and analytical parameters on the isolation, stability, and quantification of circulating miRNAs. By using quantitative stem-loop RT-PCR, we compared the relative efficiencies of four miRNA isolation systems and different storage conditions. The effect of the data normalization procedure on the quantification of circulating miRNA levels in plasma from 30 healthy individuals and 30 patients with non–small cell lung carcinoma was estimated by measuring endogenous hsa-miR-21 and hsa-miR-16 and exogenous cel-miR-39 that was spiked in all samples at the same concentration. Silica column–based RNA extraction methods are more effective and reliable with respect to TRIzol LS. Endogenous circulating miRNA levels are unstable when plasma is stored at 4°C, and samples should be kept at −70°C, where the extracted miRNAs remain stable for up to 1 year. When normalization is based on combined endogenous and exogenous control miRNAs, differences in miRNA recovery and differences in cDNA synthesis between samples are compensated. Using this normalization procedure and hsa-miR-21 as a biomarker, we could clearly discriminate healthy individuals from patients with cancer. Experimental handling and the use of exogenous and endogenous controls for normalization are critical for the reliable quantification of circulating miRNA levels in plasma. CME Accreditation Statement: This activity (“JMD 2013 CME Program in Molecular Diagnostics”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity (“JMD 2013 CME Program in Molecular Diagnostics”) for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. CME Accreditation Statement: This activity (“JMD 2013 CME Program in Molecular Diagnostics”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity (“JMD 2013 CME Program in Molecular Diagnostics”) for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. miRNAs are a class of small, endogenous, noncoding, single-strand RNAs that can negatively regulate gene expression by binding to specific complementary sites at the 3′ untranslated region of target mRNAs, causing translational repression or transcript degradation. miRNAs are involved in many important biological processes, such as cell proliferation, differentiation, and apoptosis.1Cortez M.A. Bueso-Ramos C. Ferdin J. Lopez-Berestein G. Sood A.K. Calin G.A. MicroRNAs in body fluids: the mix of hormones and biomarkers.Nat Rev Clin Oncol. 2011; 8: 467-477Crossref PubMed Scopus (1131) Google Scholar Because half of the miRNA genes in humans are located at fragile chromosomal regions that display deletions, amplifications, or translocations, aberrant expression of miRNAs occurs frequently, and thus miRNAs may act as oncogenes or tumor suppressor genes, depending on their target genes.2Sotiropoulou G. Pampalakis G. Lianidou E. Mourelatos Z. Emerging roles of microRNAs as molecular switches in the integrated circuit of the cancer cell.RNA. 2009; 15: 1443-1461Crossref PubMed Scopus (140) Google Scholar, 3Croce C.M. Causes and consequences of microRNA dysregulation in cancer.Nat Rev Genet. 2009; 10: 704-714Crossref PubMed Scopus (2588) Google Scholar Deregulation of miRNA expression levels has been detected in many human tumor types and plays a critical role in cancer pathogenesis.4Calin G.A. Croce C.M. MicroRNA signatures in human cancers.Nat Rev Cancer. 2006; 6: 857-866Crossref PubMed Scopus (6616) Google Scholar, 5Kasinski A.L. Slack F.J. Epigenetics and genetics. MicroRNAs en route to the clinic: progress in validating and targeting microRNAs for cancer therapy.Nat Rev Cancer. 2011; 11: 849-864Crossref PubMed Scopus (800) Google Scholar Despite the fact that there are <2000 human miRNAs, recent studies estimate that more than one-third of the cellular transcriptome is regulated by miRNAs. Numerous recent studies have shown that miRNAs are rapidly released from tissues into the circulation in many pathologic conditions.1Cortez M.A. Bueso-Ramos C. Ferdin J. Lopez-Berestein G. Sood A.K. Calin G.A. MicroRNAs in body fluids: the mix of hormones and biomarkers.Nat Rev Clin Oncol. 2011; 8: 467-477Crossref PubMed Scopus (1131) Google Scholar The presence of circulating miRNAs in peripheral blood in a highly stable manner that is protected from degradation conditions and factors such as boiling, extreme pH values, and endogenous RNase activity has been clearly demonstrated.6Chen X. Ba Y. Ma L. Cai X. Yin Y. Wang K. Guo J. Zhang Y. Chen J. Guo X. Li Q. Li X. Wang W. Zhang Y. Wang J. Jiang X. Xiang Y. Xu C. Zheng P. Zhang J. Li R. Zhang H. Shang X. Gong T. Ning G. Wang J. Zen K. Zhang J. Zhang C.Y. Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases.Cell Res. 2008; 18: 997-1006Crossref PubMed Scopus (3768) Google Scholar, 7Mitchell P.S. Parkin R.K. Kroh E.M. Fritz B.R. Wyman S.K. Pogosova-Agadjanyan E.L. Peterson A. Noteboom J. O'Briant K.C. Allen A. Lin D.W. Urban N. Drescher C.W. Knudsen B.S. Stirewalt D.L. Gentleman R. Vessella R.L. Nelson P.S. Martin D.B. Tewari M. Circulating microRNAs as stable blood-based markers for cancer detection.Proc Natl Acad Sci U S A. 2008; 105: 10513-10518Crossref PubMed Scopus (6440) Google Scholar This relatively high stability of miRNAs in biofluids such as plasma, serum, urine, and saliva and the ability of miRNA expression profiles to accurately classify discrete tissue types and disease states have positioned miRNAs as promising noninvasive new biomarkers for a wide range of diagnostic applications. In blood, miRNAs can circulate withstanding degradation through their inclusion in microvesicles or exosomes that are secreted from cells or by binding to high-density lipoproteins8Vickers K.C. Palmisano B.T. Shoucri B.M. Shamburek R.D. Remaley A.T. MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins.Nat Cell Biol. 2011; 13: 423-433Crossref PubMed Scopus (2112) Google Scholar or to the argonaute 2 protein complex.9Arroyo J.D. Chevillet J.R. Kroh E.M. Ruf I.K. Pritchard C.C. Gibson D.F. Mitchell P.S. Bennett C.F. Pogosova-Agadjanyan E.L. Stirewalt D.L. Tait J.F. Tewari M. Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma.Proc Natl Acad Sci U S A. 2011; 108: 5003-5008Crossref PubMed Scopus (2509) Google Scholar This, combined with the strong link between their deregulation and cancer development and progression, highlights the potential of these molecules as promising noninvasive biomarkers. Actually, to date, many research reports have demonstrated that circulating miRNA profiles can reflect physiologic and pathologic conditions.6Chen X. Ba Y. Ma L. Cai X. Yin Y. Wang K. Guo J. Zhang Y. Chen J. Guo X. Li Q. Li X. Wang W. Zhang Y. Wang J. Jiang X. Xiang Y. Xu C. Zheng P. Zhang J. Li R. Zhang H. Shang X. Gong T. Ning G. Wang J. Zen K. Zhang J. Zhang C.Y. Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases.Cell Res. 2008; 18: 997-1006Crossref PubMed Scopus (3768) Google Scholar, 7Mitchell P.S. Parkin R.K. Kroh E.M. Fritz B.R. Wyman S.K. Pogosova-Agadjanyan E.L. Peterson A. Noteboom J. O'Briant K.C. Allen A. Lin D.W. Urban N. Drescher C.W. Knudsen B.S. Stirewalt D.L. Gentleman R. Vessella R.L. Nelson P.S. Martin D.B. Tewari M. Circulating microRNAs as stable blood-based markers for cancer detection.Proc Natl Acad Sci U S A. 2008; 105: 10513-10518Crossref PubMed Scopus (6440) Google Scholar, 10Sun Y. Zhang K. Fan G. Li J. Identification of circulating microRNAs as biomarkers in cancers: what have we got?.Clin Chem Lab Med. 2012; 50: 2121-2126Crossref PubMed Scopus (25) Google Scholar, 11Reid G. Kirschner M.B. van Zandwijk N. Circulating microRNAs: association with disease and potential use as biomarkers.Crit Rev Oncol Hematol. 2011; 80: 193-208Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar In a variety of solid tumors and hematopoietic malignancies, cell-free miRNAs displayed diagnostic and prognostic value, enhancing the challenge of exploiting circulating miRNAs as molecular biomarkers for early tumor detection, diagnosis, and prognosis as well as monitoring of therapeutic response.12Redis R.S. Berindan-Neagoe I. Pop V.I. Calin G.A. Non-coding RNAs as theranostics in human cancers.J Cell Biochem. 2012; 113: 1451-1459PubMed Google Scholar, 13Mostert B. Sieuwerts A.M. Martens J.W. Sleijfer S. Diagnostic applications of cell-free and circulating tumor cell-associated miRNAs in cancer patients.Expert Rev Mol Diagn. 2011; 11: 259-275PubMed Google Scholar This growing interest in developing circulating miRNAs as blood-based biomarkers necessitates very careful consideration of the effects of various preanalytical and analytical parameters on their measurements. Preanalytical parameters, such as sample handling and storage conditions before processing, play a significant role in the reliability and reproducibility of circulating miRNA quantification.14McDonald J.S. Milosevic D. Reddi H.V. Grebe S.K. Algeciras-Schimnich A. Analysis of circulating microRNA: preanalytical and analytical challenges.Clin Chem. 2011; 57: 833-840Crossref PubMed Scopus (516) Google Scholar, 15Becker N. Lockwood C.M. Pre-analytical variables in miRNA analysis.Clin Biochem. 2013; 46: 861-868Crossref PubMed Scopus (62) Google Scholar, 16Zampetaki A. Mayr M. Analytical challenges and technical limitations in assessing circulating miRNAs.Thromb Haemost. 2012; 108: 592-598Crossref PubMed Scopus (103) Google Scholar, 17de Planell-Saguer M. Rodicio M.C. Analytical aspects of microRNA in diagnostics: a review.Anal Chim Acta. 2011; 699: 134-152Crossref PubMed Scopus (203) Google Scholar Besides, a technical hurdle to the study of miRNA expression is the ability to reliably and efficiently extract miRNAs from biological samples because of their small size and their attachment to lipids and proteins. Recently, several commercial extraction kits have become available that seek to optimize the extraction of small RNAs, either in conjunction with full-length total RNA or as a fraction enriched for small RNAs, and exogenous synthetic miRNAs have been proposed as external controls to normalize sample-to-sample variations in RNA isolation procedures.7Mitchell P.S. Parkin R.K. Kroh E.M. Fritz B.R. Wyman S.K. Pogosova-Agadjanyan E.L. Peterson A. Noteboom J. O'Briant K.C. Allen A. Lin D.W. Urban N. Drescher C.W. Knudsen B.S. Stirewalt D.L. Gentleman R. Vessella R.L. Nelson P.S. Martin D.B. Tewari M. Circulating microRNAs as stable blood-based markers for cancer detection.Proc Natl Acad Sci U S A. 2008; 105: 10513-10518Crossref PubMed Scopus (6440) Google Scholar, 18Zhu W. Qin W. Atasoy U. Sauter E.R. Circulating microRNAs in breast cancer and healthy subjects.BMC Res Notes. 2009; 2: 89Crossref PubMed Scopus (325) Google Scholar In conclusion, it is important to establish standardized protocols for blood collection, sample storage conditions, inclusion of exogenous and endogenous miRNA controls for each clinical sample, and standardized calculations for normalization of the results to ensure the reproducible and accurate quantification of circulating miRNA levels so that miRNA analysis can be implemented in the clinical laboratory setting.19Hastings M.L. Palma J. Duelli D.M. Sensitive PCR-based quantitation of cell-free circulating microRNAs.Methods. 2012; 58: 144-150Crossref PubMed Scopus (30) Google Scholar, 20Tsongalis G.J. Calin G. Cordelier P. Croce C. Monzon F. Szafranska-Schwarzbach: MicroRNA analysis: is it ready for prime time?.Clin Chem. 2013; 59: 343-347Crossref PubMed Scopus (9) Google Scholar The aim of this study was to evaluate the effects of preanalytical and analytical parameters on the isolation, stability, and quantification of circulating miRNAs in plasma. We compared the relative efficiencies of four different miRNA isolation systems and evaluated the effects of different storage conditions and storage periods on the quantification of plasma miRNA levels. Moreover, we evaluated the use of exogenous synthetic miRNAs as external controls for normalization of sample-to-sample variations in RNA isolation procedures and the use of endogenous miRNAs as normalizers for miRNA quantification. For the evaluation of four different protocols for the extraction of circulating miRNAs, whole blood samples were collected from healthy individuals in EDTA-containing tubes (BD Vacutainer; Becton Dickinson and Company, Franklin Lakes, NJ) and subjected to centrifugation at 2000 × g for 10 minutes at room temperature. The upper plasma layer was immediately pooled after centrifugation and was aliquoted into RNase-free tubes. These aliquots were used on the same day for the extraction of circulating miRNAs by four different extraction protocols. The same practice was followed for studying the stability of circulating miRNAs in plasma under different storage conditions. In this case, different aliquots of pooled plasma samples from healthy individuals were stored at different temperatures for different periods until the extraction of circulating miRNAs. One plasma aliquot was immediately extracted (baseline control, 0 hour), whereas the remaining aliquots were stored for 24 hours, 48 hours, 1 month, and 4 months at 4°C, −20°C, and −70°C until miRNA extraction. These periods were selected to represent short-term sample processing in a clinical laboratory and long-term sample storage for future processing. Moreover, we used plasma samples from 30 healthy individuals and 30 patients with non–small cell lung cancer (NSCLC) to evaluate the effect of different normalization procedures on the quantification of circulating miRNA levels in plasma. In all cases, a synthetic miRNA [Caenorhabditis elegans cel-miR-39 (5′-UCACCGGGUGUAAAUCAGCUUG-3′); miScript miRNA mimic (Qiagen Inc., Valencia, CA)] was added to all the plasma aliquots as an exogenous miRNA spiked-in control after addition of the denaturating solution to allow for normalization of sample-to-sample variation in the RNA isolation procedure. Moreover, mature hsa-miR-21 (5′-UAGCUUAUCAGACUGAUGUUGA-3′) and mature hsa-miR-16 (5′-UAGCAGCACGUAAAUAUUGGCG-3′) were used as endogenous miRNA controls. Before starting the miRNA extraction procedure, pooled plasma samples were subjected to a second centrifugation at 12,000 × g for 15 minutes at 4°C to remove all cellular debris. Subsequently, 25 fmol of the exogenous miRNA spiked-in control (cel-miR-39) was added to all the plasma aliquots after addition of the denaturating solution to allow for normalization of sample-to-sample variation in the RNA isolation procedure. Thereafter, isolation of plasma miRNAs was performed according to the manufacturer's instructions for each of the examined extraction protocols: i) a standard liquid-liquid extraction protocol using TRIzol LS (Invitrogen, Life Sciences, Carlsbad, CA) that is used for the isolation of total RNA: 250 μL of plasma and 750 μL of denaturing solution, ii) miRNeasy mini kit (Qiagen GmbH, Hilden, Germany): 200 μL of plasma and 1 mL of QIAzol lysis reagent, iii) mirVana PARIS kit (Ambion Inc., Life Sciences, Austin, TX): 200 μL of plasma and 200 μL of denaturing solution, and iv) miRNA purification kit (Norgen Biotek Corp., Thorold, ON, Canada): 1 mL of plasma and 1.5 mL of lysis solution. In all cases, cDNA was synthesized using the TaqMan miRNA reverse transcription kit and miRNA-specific stem-loop primers (both from Applied Biosystems, Life Sciences, Foster City, CA) in 15 μL of total volume reaction. Each reaction consisted of 5 μL of eluted plasma miRNAs and 10 μL of master mix [4.16 μL of nuclease-free H2O, 3 μL of TaqMan miRNA reverse transcription–specific primer, 1.5 μL of reverse transcription buffer, 0.19 μL of 20 U/μL RNase inhibitor, 0.15 μL of 100 mmol/L dNTPs, and 1 μL of 50 U/μL MultiScribe Reverse Transcriptase (Applied Biosystems, Life Sciences)]. Reverse transcription reaction mixture was incubated at 16°C for 30 minutes, at 42°C for 30 minutes, and at 85°C for 5 minutes and then was held at 4°C. A no–reverse transcription negative control was included in each experiment to ensure that the PCR products were not due to contamination by genomic DNA. The negative control produced no detectable signal in any of the experiments. Circulating miRNA levels were quantified by using TaqMan miRNA assays (Applied Biosystems, Life Sciences), according to the manufacturer's protocols. Quantitative RT-PCR was performed in a final volume of 10 μL containing 2 μL of cDNA template, 2 μL of nuclease-free water, 1 μL of 20× primer/probe mix from the TaqMan miRNA assay, and 5 μL of 2× TaqMan universal PCR master mix (Applied Biosystems, Life Sciences). All the reactions were run in triplicate using the LightCycler 2.0 real-time PCR instrument (Roche Applied Science, Indianapolis, IN). The reaction mixture was incubated at 95°C for 10 minutes, followed by 45 cycles of 95°C for 15 seconds and 60°C for 1 minute. Quantification cycle (Cq) values were calculated using LightCycler software version 4.05 (Roche Applied Science). Relative quantification was based on the ΔΔCq method as described by Livak and Schmittgen.21Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (124358) Google Scholar Statistical analysis was performed using the SPSS statistical package (version 21; SPSS Inc., Chicago, IL). Differences in mean Cq values for endogenous and exogenous miRNAs according to the extraction method used were estimated by the U-test. The same statistical test was also used for the evaluation of differences in mean Cq values of extracted miRNAs according to the different storage periods. Circulating miRNA stability concerning different storage times and different temperatures was calculated by the repeated-measures analysis of variance test. A P < 0.05 was considered statistically significant. We evaluated the efficacy and analytical performance of all these different protocols and experimental conditions by quantifying the levels of the spiked-in exogenous control miRNA cel-miR-39. Recovery of cel-miR-39 in each case was estimated with respect to the levels of an equivalent amount of cel-miR-39 copies that we added to the eluted RNA after the extraction step in each case (representing 100% recovery), when we evaluated the different extraction protocols. In parallel, in all the samples, endogenous hsa-miR-21 and hsa-miR-16 levels were also quantified by quantitative RT-PCR (RT-qPCR). We first evaluated the efficacy of TRIzol LS reagent, a widely applied method for the isolation of total RNA, for the extraction of circulating miRNAs. Therefore, we first tried to optimize the TRIzol LS protocol for isolating circulating miRNAs from plasma samples with respect to the percentage yield by examining the effects of the initial plasma volume, temperature, and incubation time of the RNA precipitation step. We used 200, 250, 300, and 500 μL of plasma and performed the precipitation step of RNA at room temperature for 10 minutes (according to the manufacturer's recommendations) or overnight at −20°C. We evaluated the efficacy and analytical performance of these different conditions by quantifying the levels of a spiked-in exogenous miRNA, cel-miR-39, that was added as an exogenous control. Recovery of cel-miR-39 in each case was estimated with respect to the levels of an equivalent amount of cel-miR-39 copies that we added to the eluted RNA after the extraction step in each case (representing 100% recovery). In parallel, in all these samples, the endogenous hsa-miR-21 was also quantified by RT-qPCR. According to the present results, better recovery for cel-miR-39 (exogenous control) and hsa-miR-21 (endogenous miRNA) was achieved when starting from 200 μL of plasma and when the RNA precipitation step was performed at room temperature for 10 minutes (Figure 1). To compare the efficacy and analytical performance of the four different commercially available kits, we used the manufacturer-suggested plasma volume and instructions except for TRIzol LS reagent, where we used 200 μL of plasma, which we found optimal as previously shown. All the experiments were performed in triplicate for the whole analytical procedure. These different isolation approaches were compared with respect to the percentage yield for cel-miR-39 used as an exogenous control and the expression levels of hsa-miR-21 used as an endogenous miRNA, as described previously herein. As can be seen in Figure 2, by using TRIzol LS reagent, we got lower percentage yields from plasma samples for cel-miR-39 and hsa-miR-21 because the recovery of spiked-in cel-miR-39 by using TRIzol LS was significantly lower compared with the mirVana PARIS kit (P = 0.039). Taking into account the amount of sample required in each case, we preferred to proceed further with the mirVana PARIS kit. We investigated the stability of circulating miRNAs in plasma at different storage temperatures and for different periods by measuring the levels of the exogenous control (cel-miR-39) and two endogenous miRNAs (hsa-miR-21 and hsa-miR-16) using RT-qPCR. All the experiments were performed in triplicate for the whole analytical procedure. The same amount of exogenous control (cel-miR-39) was spiked-in after thawing in all the plasma samples to evaluate whether the detected differences in recovery rates were due to the storage conditions only and not to errors of the extraction procedure used. The results clearly indicate that endogenous circulating miRNA levels are unstable when plasma specimens are stored at 4°C after 24 hours, 48 hours, 1 month, and 4 months because there was an increase in Cq values in all cases (Figure 3). Storage of plasma samples at −20°C or −70°C for 4 months led to increases of approximately 4.0 and 3.0 in Cq values, respectively, indicating that for long-term maintenance of plasma samples, −20°C or −70°C temperature conditions are more appropriate (P < 0.05). In parallel, the levels of exogenous cel-miR-39 for each extraction on different days seem to be almost equal, thus eliminating the possibility that the increased Cq values are attributed to sample-to-sample variation during their extraction (Figure 3). Thus, these differences in Cq values for the endogenous miRNAs that are statistically significant intimate the degradation of miRNAs during long-term plasma storage. Additionally, we evaluated the stability of extracted miRNAs at −70°C for a storage period of up to 12 months by quantifying cel-miR-39 levels after 2, 6, 8, 9, and 12 months. These results indicate that extracted miRNAs are very stable when stored at −70°C for 1 year because slight differences in Cq values were not significant (P > 0.05) (Figure 4). We first isolated circulating miRNAs from plasma samples from 30 healthy individuals and 30 patients with NSCLC by using the mirVana PARIS kit. We evaluated the effect of different normalization procedures on the quantification of circulating miRNAs in these plasma samples by measuring endogenous hsa-miR-21 and hsa-miR-16 levels and levels of the exogenous control cel-miR-39 that was spiked in all the samples at the same concentration. The concentration of hsa-miR-21 in plasma was quantified by normalizing with respect to i) hsa-miR-16, used as an endogenous control of stable expression (Figure 5A); ii) exogenous control cel-miR-39 (Figure 5B); and iii) a combination of the two, where the expression levels of both hsa-miR-21 and the endogenous control hsa-miR-16 were normalized with respect to the exogenous control cel-miR-39, and then ΔCq was estimated (Figure 5C). These results clearly suggest that when normalization is based on a combination of an endogenous and an exogenous control miRNA, differences in the recovery of miRNAs from plasma and differences in cDNA synthesis between samples are compensated. By using this normalization procedure, we could clearly discriminate controls from patients with cancer when hsa-miR-21 was used as a biomarker. Circulating miRNAs are promising biomarkers for various diseases, especially cancer. However, their establishment in the clinical laboratory setting requires highly sensitive, reproducible, reliable, and robust assays enabling their accurate quantification in plasma and serum. To achieve this goal, a variety of preanalytical and analytical parameters that can influence their isolation and stability should be checked. Moreover, there is a great demand for robust and reliable approaches for the normalization of miRNA quantitative data. Herein we evaluated the effect of preanalytical and analytical parameters on the isolation, stability, and quantification of circulating miRNAs in plasma. We compared the relative efficiencies of four different miRNA isolation systems and evaluated the effect of different storage conditions and storage times on the quantification of plasma miRNA levels and evaluated the use of exogenous synthetic miRNAs as external controls for data normalization of sample-to-sample variations in RNA isolation procedures and the use of endogenous miRNAs as normalizers for miRNA quantification. Recently, a variety of studies have addressed some important steps for the establishment of robust strategies for blood-based miRNA profiling toward its implementation in routine handling for diagnostic purposes. It has already been reported that preanalytical issues, such as endogenous serum factors that are copurified with miRNAs and anticoagulant agents used during collection, can seriously influence the quantification of circulating miRNA levels22Kim D.J. Linnstaedt S. Palma J. Park J.C. Ntrivalas E. Kwak-Kim J.Y. Gilman-Sachs A. Beaman K. Hastings M.L. Martin J.N. Duelli D.M. Plasma components affect accuracy of circulating cancer-related microRNA quantitation.J Mol Diagn. 2012; 14: 71-80Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar and that sample hemolysis alters miRNA content in plasma or serum23Kirschner M.B. Kao S.C. Edelman J.J. Armstrong N.J. Vallely M.P. van Zandwijk N. Reid G. Haemolysis during sample preparation alters microRNA content of plasma.PLoS One. 2011; 6: e24145Crossref PubMed Scopus (411) Google Scholar; however, the extraction method used and the underlying source have been found to result in dissimilar miRNA expression profiles.24Gaarz A. Debey-Pascher S. Classen S. Eggle D. Gathof B. Chen J. Fan J.B. Voss T. Schultze J.L. Staratschek-Jox A. Bead array-based microrna expression profiling of peripheral blood and the impact of different RNA isolation approaches.J Mol Diagn. 2010; 12: 335-344Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar Li and Kowdley 25Li Y. Kowdley K.V. Method for microRNA isolation from clinical serum samples.Anal Biochem. 2012; 431: 69-75Crossref PubMed Scopus (97) Google Scholar evaluated three commonly used commercial miRNA isolation kits for the best performance by comparing RNA quality and yield. In a very recent study, Callari et al26Callari M. Tiberio P. De Cecco L. Cavadini E. Dugo M. Ghimenti C. Daidone M.G. Canevari S. Appierto V. Feasibility of circulating miRNA microarray analysis from archival plasma samples.Anal Biochem. 2013; 437: 123-125Crossref PubMed Scopus (23) Google Scholar investigated whether circulating miRNAs can be reliably analyzed by microarrays from archival plasma samples. Moreover, the miRNA quantification strategy followed and especially the data normalization procedure have a significant effect on the reliability of miRNA quantification.27Redshaw N. Wilkes T. Whale A. Cowen S. Huggett J. Foy C.A. A comparison of miRNA isolation and RT-qPCR technologies and their effects on quantification accuracy and repeatability.Biotechniques. 2013; 54: 155-164Crossref PubMed Scopus (99) Google Scholar, 28Kroh E.M. Parkin R.K. Mitchell P.S. Tewari M. Analysis of circulating microRNA biomarkers in plasma and serum using quantitative reverse transcription-PCR (qRT-PCR).Methods. 2010; 50: 298-301Crossref PubMed Scopus (968) Google Scholar The small size of miRNAs and their binding on lipids and proteins while in circulation in body fluids affects their reproducible recovery from plasma samples. These results on the analytical performance of three different column-based isolation protocols and that of TRIzol LS reagent indicated that column-based protocols are more effective and reliable and display a remarkable reproducibility concerning the isolation of cell-free miRNAs. By using TRIzol LS reagent based on the phenol:chloroform extraction, we could not achieve the same efficacy for endogenous and exogenous miRNAs despite the optimization steps that we performed before the comparison study. Among the commercially available kits that we evaluated, the mirVana PARIS kit was more reproducible with a higher miRNA yield. These results were consistent with previous findings by McDonald et al,14McDonald J.S. Milosevic D. Reddi H.V. Grebe S.K. Algeciras-Schimnich A. Analysis of circulating microRNA: preanalytical and analytical challenges.Clin Chem. 2011; 57: 833-840Crossref PubMed Scopus (516) Google Scholar who reported that the mirVana PARIS kit was found to be the best-performing column-based kit for isolating RNAs among four miRNA extraction kits compared (two of them were the mirVana PARIS kit from Ambion Inc. and the miRNeasy mini kit from Qiagen Inc., which were also used in the present study). However, the present results were in contrast with previous findings from the same group who also reported that TRIzol LS extraction matches or even surpasses the performance of the mirVana PARIS kit.14McDonald J.S. Milosevic D. Reddi H.V. Grebe S.K. Algeciras-Schimnich A. Analysis of circulating microRNA: preanalytical and analytical challenges.Clin Chem. 2011; 57: 833-840Crossref PubMed Scopus (516) Google Scholar Sample storage conditions can seriously affect the accuracy and reliability of analytical results. We estimated the stability of circulating miRNAs in identical plasma samples under different temperature conditions for different periods. These results indicate that for the accurate quantification of cell-free miRNAs, the isolation process should be conducted within 48 hours of sample collection when keeping plasma samples at −20°C or −70°C, whereas storage at 4°C, even for 24 hours, leads to a significant reduction in circulating miRNA levels. When long-term storage of plasma samples is required, then −70°C rather −20°C should be used to avoid extensive miRNA degradation. Similar results have been presented by Grasedieck et al29Grasedieck S. Schöler N. Bommer M. Niess J.H. Tumani H. Rouhi A. Bloehdorn J. Liebisch P. Mertens D. Döhner H. Buske C. Langer C. Kuchenbauer F. Impact of serum storage conditions on microRNA stability.Leukemia. 2012; 26: 2414-2416Crossref PubMed Scopus (121) Google Scholar with respect to the impact of serum storage conditions on miRNA stability. We also evaluated the stability of miRNAs in the extraction buffer because this is very useful in the clinical laboratory setting, especially where novel findings suggest testing additional miRNAs in archived miRNA extraction samples. The results indicated that extracted miRNAs can remain stable for ≥1 year at −70°C. However, some studies report high instability and significant degradation of miRNAs 3 days after their isolation,30Bravo V. Rosero S. Ricordi C. Pastori R.L. Instability of miRNA and cDNAs derivatives in RNA preparations.Biochem Biophys Res Commun. 2007; 353: 1052-1055Crossref PubMed Scopus (54) Google Scholar whereas other studies report high stability of isolated miRNAs over approximately 10 months.31Mraz M. Malinova K. Mayer J. Pospisilova S. MicroRNA isolation and stability in stored RNA samples.Biochem Biophys Res Commun. 2009; 390: 1-4Crossref PubMed Scopus (165) Google Scholar One explanation could be the use of different elution buffers for miRNA storage in each case. The present results indicate that the isolated miRNA fraction from plasma samples using the mirVana PARIS kit can be available for a long time at deep freeze. Finally, we assessed the use of endogenous and exogenous controls for qPCR data normalization. This is a very critical issue to ensure the robustness of data owing to all the previously referred technical variations. In the field of circulating miRNAs, there is no generally accepted standard procedure for data normalization. One choice that is almost generally accepted now is the spiking of synthetic miRNAs into plasma samples before the extraction process and the measurement of their levels by RT-qPCR as normalizers.7Mitchell P.S. Parkin R.K. Kroh E.M. Fritz B.R. Wyman S.K. Pogosova-Agadjanyan E.L. Peterson A. Noteboom J. O'Briant K.C. Allen A. Lin D.W. Urban N. Drescher C.W. Knudsen B.S. Stirewalt D.L. Gentleman R. Vessella R.L. Nelson P.S. Martin D.B. Tewari M. Circulating microRNAs as stable blood-based markers for cancer detection.Proc Natl Acad Sci U S A. 2008; 105: 10513-10518Crossref PubMed Scopus (6440) Google Scholar, 28Kroh E.M. Parkin R.K. Mitchell P.S. Tewari M. Analysis of circulating microRNA biomarkers in plasma and serum using quantitative reverse transcription-PCR (qRT-PCR).Methods. 2010; 50: 298-301Crossref PubMed Scopus (968) Google Scholar Alternatively, endogenous miRNAs with low variability among the samples have also been used as normalizers by many research groups.18Zhu W. Qin W. Atasoy U. Sauter E.R. Circulating microRNAs in breast cancer and healthy subjects.BMC Res Notes. 2009; 2: 89Crossref PubMed Scopus (325) Google Scholar, 32Roth C. Rack B. Müller V. Janni W. Pantel K. Schwarzenbach H. Circulating microRNAs as blood-based markers for patients with primary and metastatic breast cancer.Breast Cancer Res. 2010; 12: R90Crossref PubMed Scopus (351) Google Scholar In the present study, we evaluated the effect of the data normalization procedure on the quantification of circulating miRNA levels in plasma from 30 healthy individuals and 30 patients with NSCLC by measuring levels of endogenous hsa-miR-21 and hsa-miR-16 and exogenous cel-miR-39 that was spiked in all samples at the same concentration. These data suggest that when normalization is based on a combination of an endogenous and an exogenous control miRNA, differences in miRNA recovery and differences in cDNA synthesis between samples are compensated. Using this normalization procedure and hsa-miR-21 as a biomarker, we could clearly discriminate healthy individuals from patients with cancer. It has been clearly shown that the expression of hsa-miR-21 can be differentiated between NSCLC and controls for fresh frozen tissue pairs33Markou A. Tsaroucha E.G. Kaklamanis L. Fotinou M. Georgoulias V. Lianidou E.S. Prognostic value of mature microRNA-21 and microRNA-205 overexpression in non-small cell lung cancer by quantitative real-time RT-PCR.Clin Chem. 2008; 54: 1696-1704Crossref PubMed Scopus (400) Google Scholar, 34Markou A. Sourvinou I. Vorkas P.A. Yousef G.M. Lianidou E.S. Clinical evaluation of microRNA expression profiling in non small cell lung cancer.Lung Cancer. 2013; 81: 388-396Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar and plasma.34Markou A. Sourvinou I. Vorkas P.A. Yousef G.M. Lianidou E.S. Clinical evaluation of microRNA expression profiling in non small cell lung cancer.Lung Cancer. 2013; 81: 388-396Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 35Saito M. Schetter A.J. Mollerup S. Kohno T. Skaug V. Bowman E.D. Mathé E.A. Takenoshita S. Yokota J. Haugen A. Harris C.C. The association of microRNA expression with prognosis and progression in early-stage, non-small cell lung adenocarcinoma: a retrospective analysis of three cohorts.Clin Cancer Res. 2011; 17: 1875-1882Crossref PubMed Scopus (170) Google Scholar, 36Heegaard N.H. Schetter A.J. Welsh J.A. Yoneda M. Bowman E.D. Harris C.C. Circulating micro-RNA expression profiles in early stage nonsmall cell lung cancer.Int J Cancer. 2012; 130: 1378-1386Crossref PubMed Scopus (249) Google Scholar There is no gold standard to normalize Cq values and, thus, determine the actual quantitative level of these expression measurements and the magnitude of the difference between cancer and healthy plasma samples. This has led to many different approaches for miRNA normalization in plasma samples. We strongly believe that the proposed approach combines the advantages of an endogenous control, reassuring us of the sample quality, and an exogenous control, helping correct for differences in recovery rates between different samples. These results are in agreement with those of Blondal et al,37Blondal T. Jensby Nielsen S. Baker A. Andreasen D. Mouritzen P. Wrang Teilum M. Dahlsveen IK: Assessing sample and miRNA profile quality in serum and plasma or other biofluids.Methods. 2013; 59: S1-S16Crossref PubMed Scopus (487) Google Scholar who also reported a similar normalization approach for high-quality data from miRNA expression profiling studies. Consistent with previous studies, our results confirm that preanalytical and analytical variables can seriously affect the reliability and reproducibility of circulating miRNA quantification. In particular, the use of exogenous and endogenous controls for normalization is critical for the reliable quantification of circulating miRNA levels in plasma. These factors should be taken into account to translate analysis of circulating miRNAs in body fluids into validated clinical tests in a routine clinical setting." @default.
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