Improved Detection of Circulating miRNAs in Serum and Plasma Following Rapid Heat/Freeze Cycling.
ABSTRACT: BACKGROUND:The measurement of circulating miRNAs has proven to be a powerful biomarker tool for several disease processes. Current protocols for the detection of miRNAs usually involve an RNA extraction step, requiring a substantial volume of patient serum or plasma to obtain sufficient input material. OBJECTIVE:Here, we describe a novel methodology that allows detection of a large number of miRNAs from a small volume of serum or plasma without the need for RNA extraction. METHODS:Three ?l of serum or plasma was subjected to three cycles of high and low temperatures (heat/freeze cycles) followed by miRNA arrays. RESULTS:Our results indicate that miRNA detection following this process is highly reproducible when comparing multiple samples from the same subject. Moreover, this protocol increases the reproducibility of miRNA detection in samples that were previously subjected to multiple freeze-thaw cycles. Importantly, the detection of miRNAs from serum vs. plasma following heat/freeze cycling are highly comparable, indicating that this heat/freeze process effectively eliminates differences in detection between serum and plasma samples that have been reported using other sample preparation methodologies. CONCLUSION:We propose that this method is a potent alternative to current RNA extraction protocols, substantially reducing the amount of sample necessary for miRNA detection while simultaneously improving miRNA detection and reproducibility.
Project description:BACKGROUND AND AIM:The potential of microRNAs (miRNA) as non-invasive diagnostic, prognostic, and predictive biomarkers, as well as therapeutic targets, has recently been recognized. Previous studies have highlighted the importance of consistency in the methodology used, but to our knowledge, no study has described the methodology of sample preparation and storage systematically with respect to miRNAs as blood biomarkers. The aim of this study was to investigate the stability of miRNAs in blood under various relevant clinical and research conditions: different collection tubes, storage at different temperatures, physical disturbance, as well as serial freeze-thaw cycles. METHODS:Blood samples were collected from 12 healthy donors into different collection tubes containing anticoagulants, including EDTA, citrate and lithium-heparin, as well as into serum collection tubes. MiRNA stability was evaluated by measuring expression changes of miR-1, miR-21 and miR-29b at different conditions: varying processing time of whole blood (up to 72 hours (h)), long-term storage (9 months at -80°C), physical disturbance (1 and 8 h), as well as in a series of freeze/thaw cycles (1 and 4 times). RESULTS:Different collection tubes revealed comparable concentrations of miR-1, miR-21 and miR-29b. Tubes with lithium-heparin were found unsuitable for miRNA quantification. MiRNA levels were stable for at least 24 h at room temperature in whole blood, while separated fractions did show alterations within 24 h. There were significant changes in the miR-21 and miR-29b levels after 72 h incubation of whole blood at room temperature (p<0.01 for both). Both miR-1 and miR-21 showed decreased levels after physical disturbance for 8 h in separated plasma and miR-1 in serum whole blood, while after 1 h of disturbance no changes were observed. Storage of samples at -80°C extended the miRNA stability remarkably, however, miRNA levels in long-term stored (9 months) whole blood samples were significantly changed, which is in contrast to the plasma samples, where miR-21 or miR-29b levels were found to be stable. Repetitive (n = 4) freeze-thaw cycles resulted in a significant reduction of miRNA concentration both in plasma and serum samples. CONCLUSION:This study highlights the importance of proper and systematic sample collection and preparation when measuring circulating miRNAs, e.g., in context of clinical trials. We demonstrated that the type of collection tubes, preparation, handling and storage of samples should be standardized to avoid confounding variables influencing the results.
Project description:MicroRNAs regulate gene expression at the post-transcriptional level. Differential expression of miRNAs can potentially be used as biomarkers for early diagnosis and prediction for outcomes. Failure in validation of miRNA profiles is often caused by variations in experimental parameters. In this study, the performance of five extraction kits and three RT-qPCR systems were evaluated using BioMark high-throughput platform and the effects of different experimental parameters on circulating miRNA levels were determined. Differences in the performance of extraction kits as well as varying accuracy, sensitivity and reproducibility in qPCR systems were observed. Normalisation of RT-qPCR data to spike-in controls can reduce extraction bias. However, the extent of correlation for different qPCR systems varies in different assays. At different time points, there was no significant fold change in eight of the plasma miRNAs that we evaluated. Higher level of miRNAs was detected in plasma as compared to serum of the same cohort. In summary, we demonstrated that high-throughput RT-qPCR with pre-amplification step had increased sensitivity and can be achieved with accuracy and high reproducibility through stringent experimental controls. The information provided here is useful for planning biomarker validation studies involving circulating miRNAs.
Project description:Circulating nucleic acids (CNAs) are under investigation as a liquid biopsy in cancer. However there is wide variation in blood processing and methods for isolation of circulating free DNA (cfDNA) and microRNAs (miRNAs). Here we compare the extraction efficiency and reproducibility of 4 commercially available kits for cfDNA and 3 for miRNA using spike-in of reference templates. We also compare the effects of increasing time between venepuncture and centrifugation and differential centrifugation force on recovery of CNAs. cfDNA was quantified by TaqMan qPCR and targeted deep sequencing. miRNA profiles were assessed with TaqMan low-density arrays and assays. The QIAamp(®) DNA Blood Mini and Circulating nucleic acid kits gave the highest recovery of cfDNA and efficient recovery (>90%) of a 564bp spike-in. Moreover, targeted sequencing revealed overlapping cfDNA profiles and variant depth, including detection of HER2 gene amplification, using the Ion AmpliSeq™Cancer Hotspot Panel v2. Highest yields of miRNA and the synthetic Arabidopsis thaliana miR-159a spike-in were obtained using the miRNeasy Serum/Plasma kit, with saturation above 200 µl of plasma. miRNA profiles showed significant variation with increasing time before centrifugation (p<0.001) and increasing centrifugation force, with depletion of platelet associated miRNAs, whereas cfDNA was unaffected. However, sample replicates showed excellent reproducibility on TaqMan low density arrays (? = 0.96, p<0.0001). We also successfully generated miRNA profiles for plasma samples stored > 12 years, highlighting the potential for analysis of stored sample biobanks. In the era of the liquid biopsy, standardisation of methods is required to minimise variation, particularly for miRNA.
Project description:Circulating microRNAs (ci-miRNAs) in blood have emerged as promising diagnostic, prognostic and predictive biomarkers in cancer. Many clinical studies currently incorporate studies that assess ci-miRNAs. Validation of the clinical significance of candidate biomarker miRNAs has proven to be difficult, potentially resulting from vast discrepancies in the detection methodology as well as biological variability. In the current study, the influence of several methodological factors on ci-miRNA detection was evaluated as well as short-term biological variability in patients with head and neck cancer. RNA was isolated from 124 serum and plasma samples originating from patients with head and neck cancer and healthy volunteers. The miRNA levels were measured using RT-qPCR and the influence of pre-analytical factors, different normalization strategies and temporal reproducibility was assessed. RNA carriers improved ci-miRNA detection in serum and plasma specimens. A prolonged pre-processing time correlated with an increased hemolytic index in serum samples only. Hemolysis differentially affected the detection of individual miRNAs. Optimal normalization was achieved using the averaged detection values of spike-in cel-miR-39-3p and endogenous miR-16-5p. Comparing biological replicates from patients with head and neck cancer, the intra-individual miRNA levels were relatively stable (average interval 7 days). Differences in the ci-miRNA detection methodology and limitations of currently used technologies can greatly affect the results and may explain inconsistent outcomes between studies. Prior to the implementation of ci-miRNAs as useful clinical biomarkers, further advances in the standardization of the detection methodology and reduction of technical variability are needed.
Project description:The effects of storage temperatures, repeated freeze-thaw cycles, or delays in separating plasma or serum from blood samples are largely unknown for heat shock protein 27 (HSP27). We evaluated (1) the imprecision of the HSP27 assay used in this study; (2) the in vitro stability of HSP27 in blood samples stored at 4°C for up to 6 hr with immediate and delayed serum/plasma separation from cells; and (3) the in vitro stability of HSP27 in blood samples stored at -80°C after repeated freeze-thaw cycles. The ELISA to detect HSP27 in this study showed a within-run CV of <9% and a total CV of <15%. After 4-6 hr of storage at 4°C, HSP27 concentrations remained stable when using serum tubes irrespective of sample handling, but HSP27 concentrations decreased by 25-45% when using EDTA plasma tubes. Compared with baseline HSP27, one freeze-thaw cycle had no effect on serum concentrations. However, plasma concentrations increased by 3.1-fold after one freeze-thaw cycle and by 7.3-fold after five freeze-thaw cycles. In conclusion, serum is an appropriate biological sample type for use in epidemiological and large-scale clinical studies.
Project description:Recent studies indicate that microRNA (miRNA) is contained within exosome. Here we sought to optimize the methodologies for the isolation and quantification of urinary exosomal microRNA as a prelude to biomarker discovery studies. Exosomes were isolated through ultracentrifugation and characterized by immunoelectron microscopy. To determine the RNA was confined inside exosomes, the pellet was treated with RNase before RNA isolation. The minimum urine volume, storage conditions for exosomes and exosomal miRNA was evaluated. The presence of miRNAs in patients with various kidney diseases was validated with real-time PCR. The result shows that miRNAs extracted from the exosomal fraction were resistant to RNase digestion and with high quality confirmed by agarose electrophoresis. 16 ml of urine was sufficient for miRNA isolation by absolute quantification with 4.15×10(5) copies/ul for miR-200c. Exosomes was stable at 4? 24h for shipping before stored at -80? and was stable in urine when stored at -80°C for 12 months. Exosomal miRNA was detectable despite 5 repeat freeze-thaw cycles. The detection of miRNA by quantitative PCR showed high reproducibility (>94% for intra-assay and >76% for inter-assay), high sensitivity (positive call 100% for CKD patients), broad dynamic range (8-log wide) and good linearity for quantification (R(2)>0.99). miR-29c and miR-200c showed different expression in different types of kidney disease. In summary, the presence of urinary exosomal miRNA was confirmed for patients with a diversity of chronic kidney disease. The conditions of urine collection, storage and miRNA detection determined in this study may be useful for future biomarker discovery efforts.
Project description:BACKGROUND:MicroRNAs (miRNAs) represent new and potentially informative diagnostic targets for disease detection and prognosis. However, little work exists documenting the effect of TRIzol, a common viral inactivation and nucleic acid extraction reagent, on miRNA purification. Here, we developed an optimized protocol for miRNA extraction from plasma samples by evaluating five different RNA extraction kits, TRIzol phase separation, purification additives, and initial plasma sample volume. This method was then used for downstream profiling of plasma miRNAs found in archived samples from one nonhuman primate (NHP) experimentally challenged with Ebola virus by the aerosol route. RESULTS:Comparison of real-time RT-PCR results for spiked-in and endogenous miRNA sequences determined extraction efficiencies from five different RNA purification kits. These experiments showed that 50 ?L plasma processed using the QIAGEN miRNeasy Mini Kit with 5 ?g of glycogen as a co-precipitant yielded the highest recovery of endogenous miRNAs. Using this optimized protocol, miRNAs from archived plasma samples of one rhesus macaque challenged with aerosolized Ebola virus was profiled using a targeted real-time PCR array. A total of 519 of the 752 unique miRNAs assayed were present in the plasma samples at day 0 and day 7 (time of death) post-exposure. Statistical analyses revealed 25 sequences significantly up- or down-regulated between day 0 and day 7 post infection, validating the utility of the extraction method for plasma miRNA profiling. CONCLUSIONS:This study contributes to the knowledgebase of circulating miRNA extraction methods and expands on the potential applications of cell-free miRNA profiling for diagnostics and pathogenesis studies. Specifically, we optimized an extraction protocol for miRNAs from TRIzol-inactivated plasma samples that can be used for highly pathogenic viruses.
Project description:MicroRNAs (miRNAs) have emerged as promising cancer biomarkers. However, exploiting their informative potential requires careful optimization of their detection. Here, we compared the efficiency of commonly used RNA extraction kits in miRNA recovery from cells, plasma and urine/plasma-derived exosomes, using single-gene RT-qPCR and miRNA profiling. We used increasing amounts of starting material to investigate the impact of the input material size on miRNA extraction. We showed that miRNA recovery was largely influenced by the isolation method and by the amount of input material. In particular, the miRCURY™ kit provided highly pure RNA. However, its columns poorly recovered miRNAs from limiting amounts of cells and plasma, and rapidly saturated by large RNA species and plasma components, thus impeding miRNA recovery from high input amounts. Overall, the miRNeasy® kit permitted a better miRNA detection despite a less pure extracted RNA. Nevertheless, some miRNAs were preferentially or exclusively isolated by either of the methods. Trizol® LS resulted in very low purity RNA which affected RT-qPCR efficiency. In general, miRCURY™ biofluids kit efficiently extracted miRNAs from plasma. A careful selection of the RNA isolation method and the consideration of the type and size of input material are highly recommended to avoid biased results.
Project description:Circulating microRNAs that are associated with specific diseases have garnered much attention for use in diagnostic assays. However, detection of disease-associated miRNA can be affected by several factors such as release of contaminating cellular miRNA during sample collection, variations due to amplification of transcript for detection, or controls used for normalization for accurate quantitation. We analyzed circulating miRNA in serum and plasma samples obtained concurrently from 28 patients, using a Nanostring quantitative assay platform. Total RNA concentration ranged from 32-125 ?g/ml from serum and 30-220 ?g/ml from plasma. Of 798 miRNAs, 371 miRNAs were not detected in either serum or plasma samples. 427 were detected in either serum or plasma but not both, whereas 151 miRNA were detected in both serum and plasma samples. The diversity of miRNA detected was greater in plasma than in serum samples. In serum samples, the number of detected miRNA ranged from 3 to 82 with a median of 17, whereas in plasma samples, the number of miRNA detected ranged from 25 to 221 with a median of 91. Several miRNA such as miR451a, miR 16-5p, miR-223-3p, and mir25-3p were highly abundant and differentially expressed between serum and plasma. The detection of endogenous and exogenous control miRNAs varied in serum and plasma, with higher levels observed in plasma. Gene expression stability identified candidate invariant microRNA that were highly stable across all samples, and could be used for normalization. In conclusion, there are significant differences in both the number of miRNA detected and the amount of miRNA detected between serum and plasma. Normalization using miRNA with constant expression is essential to minimize the impact of technical variations. Given the challenges involved, ideal candidates for blood based biomarkers would be those that are indifferent to type of body fluid, are detectable and can be reliably quantitated.
Project description:Growing interest in blood-borne microRNAs (miRNAs) as biomarkers has led to the introduction of a number of commercial kits for isolating small RNAs from plasma/serum. We sought to compare the efficacy of six such kits in isolating miRNAs from either whole plasma or a plasma-derived ultracentrifugation (UC) fraction from 2 healthy volunteers with some of the results being validated in 10 additional subjects. To assess the overall yield and concentration of isolated small RNAs, we measured the levels of one spiked-in and four endogenous miRNAs by quantitative reverse transcription and polymerase chain reaction (qRT-PCR). We also tested the performance of the Agilent Bioanalyzer small RNA assay with these RNA samples. Additionally, we tested the effects of hemolysis on measured miRNA levels in whole plasma and in the UC fraction. Both the efficiency of RNA isolation and the relative levels of specific miRNAs in different samples varied considerably between the tested extraction methods. Of all kits tested, the QIAGEN miRNeasy kits (Mini and Serum/Plasma kits) and the Macherey-Nagel NucleoSpin kit produced the highest RNA yields. The QIAGEN Exo kit produced lesser yields than what could be extracted from the UC fraction using the QIAGEN miRNeasy kits and the Macherey-Nagel NucleoSpin kit. Bioanalyzer results showed an average correlation of R 2?=?0.8 with endogenous miRNA qRT-PCR results, for sample concentrations >40?pg/µl. The levels of the endogenous miRNAs measured in the two volunteer samples were compared with those in a larger group of subjects (n?=?10) and found to be typical. Our comparison favors the use of the QIAGEN Serum/Plasma kit and the Macherey-Nagel NucleoSpin kit for plasma miRNA applications. Furthermore, extraction of miRNAs from the UC fraction results in higher yield than extraction from whole plasma.