Optimized microRNA purification from TRIzol-treated plasma.
ABSTRACT: 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:BACKGROUND: Describing and evaluating miRNA inventories with Next Generation Sequencing is a goal of scientists from a wide range of fields. It requires high purity, high quality, and high yield RNA extractions that do not only contain abundant ribosomal RNAs but are also enriched in miRNAs. Here we compare 6 disparate and commercially available totalRNA extraction kits for their suitability for miRNA-preparations from Gyrodactylus salaris, an important but small (500 ?m in length) monogenean pathogen of Norwegian Atlantic salmon (Salmo salar). FINDINGS: We evaluated 1 salt precipitation method (MasterPure™ Complete RNA Purification Kit, Epicentre), 2 Phenol based extraction methods (mirVana Kit, Ambion, and Trizol Plus Kit, Invitrogen), 1 paramagnetic bead extraction method (RNA Tissue kit, GeneMole) and 2 purification methods based on spin column chromatography using a proprietary resin as separation matrix (Phenol-free Total RNA Purification Kit, Amresco, and ZR MicroPrep Kit, Zymo Research). The quality of the extractions from 1, 10 and 100 individuals, respectively, was assessed in terms of totalRNA yield, RNA integrity, and smallRNA and miRNA yield. The 6 RNA extraction methods yielded considerably different total RNA extracts, with striking differences in low molecular weight RNA yield. The Phenol-free Total RNA Purification Kit (Amresco) showed the highest totalRNA yield, but the best miRNA/totalRNA ratio was obtained with the ZR MicroPrep Kit (Zymo Research). It was not possible to extract electrophoretically detectable miRNAs from Gyrodactylus salaris with the RNA Tissue Kit (GeneMole) or the Trizol Plus Kit (Invitrogen). CONCLUSIONS: We present an optimized extraction protocol for single and small numbers of Gyrodactylus salaris from infected Atlantic salmon that delivers a totalRNA yield suitable for downstream next generation sequencing analyses of miRNA. Two of the six tested totalRNA kits/methods were not suitable for the extraction of miRNAs from Gyrodactylus salaris.
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: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.
Project description:miRNAs act as important regulators of gene expression by promoting mRNA degradation or by attenuating protein translation. Since miRNAs are stably expressed in bodily fluids, there is growing interest in profiling these miRNAs, as it is minimally invasive and cost-effective as a diagnostic matrix. A technical hurdle in studying miRNA dynamics is the ability to reliably extract miRNA as small sample volumes and low RNA abundance create challenges for extraction and downstream applications. The purpose of this study was to develop a pipeline for the recovery of miRNA using small volumes of archived serum samples. The RNA was extracted employing several widely utilized RNA isolation kits/methods with and without addition of a carrier. The small RNA library preparation was carried out using Illumina TruSeq small RNA kit and sequencing was carried out using Illumina platform. A fraction of five microliters of total RNA was used for library preparation as quantification is below the detection limit. We were able to profile miRNA levels in serum from all the methods tested. We found out that addition of nucleic acid based carrier molecules had higher numbers of processed reads but it did not enhance the mapping of any miRBase annotated sequences. However, some of the extraction procedures offer certain advantages: RNA extracted by TRIzol seemed to align to the miRBase best; extractions using TRIzol with carrier yielded higher miRNA-to-small RNA ratios. Nuclease free glycogen can be carrier of choice for miRNA sequencing. Our findings illustrate that miRNA extraction and quantification is influenced by the choice of methodologies. Addition of nucleic acid- based carrier molecules during extraction procedure is not a good choice when assaying miRNA using sequencing. The careful selection of an extraction method permits the archived serum samples to become valuable resources for high-throughput applications.
Project description:There are increasing reports of plasma miRNAs as biomarkers of human disease but few standards in methodologic reporting, leading to inconsistent data. We systematically reviewed plasma miRNA studies published between July 2013-June 2014 to assess methodology. Six parameters were investigated: time to plasma extraction, methods of RNA extraction, type of miRNA, quantification, cycle threshold (Ct) setting, and methods of statistical analysis. We compared these data with a proposed standard methodologic technique. Beginning with initial screening for 380 miRNAs using microfluidic array technology and validation in an additional cohort of patients, we compared 11 miRNAs that exhibited differential expression between 16 patients with benign colorectal neoplasms (advanced adenomas) and 16 patients without any neoplasm (controls). Plasma was isolated immediately, 12, 24, 48, or 72 h following phlebotomy. miRNA was extracted using two different techniques (Trizol LS with pre-amplification or modified miRNeasy). We performed Taqman-based RT-PCR assays for the 11 miRNAs with subsequent analyses using a variable Ct setting or a fixed Ct set at 0.01, 0.03, 0.05, or 0.5. Assays were performed in duplicate by two different operators. RNU6 was the internal reference. Systematic review yielded 74 manuscripts meeting inclusion criteria. One manuscript (1.4%) documented all 6 methodological parameters, while < 5% of studies listed Ct setting. In our proposed standard technique, plasma extraction ?12 h provided consistent ?Ct. miRNeasy extraction yielded higher miRNA concentrations and fewer non-expressed miRNAs compared to Trizol LS (1/704 miRNAs [0.14%] vs 109/704 miRNAs [15%], not expressed, respectively). A fixed Ct bar setting of 0.03 yielded the most reproducible data, provided that <10% miRNA were non-expressed. There was no significant intra-operator variability. There was significant inter-operator variation using Trizol LS extraction, while this was negligible using modified miRNeasy. For standardized reporting, we recommend plasma extraction ? 12 h, using modified miRNeasy extraction and utilizing a 0.03 Ct.
Project description:BACKGROUND:microRNAs (miRNAs) are short non-coding RNAs that fine-tune gene expression. The aberrant expression of miRNAs is associated with many diseases and they have both therapeutic and biomarker potential. However, our understanding of their usefulness is dependent on the tools we have to study them. Previous studies have identified the need to optimise and standardise RNA extraction methods in order to avoid biased results. Herein, we extracted RNA from murine lung, liver and brain tissues using five commercially available total RNA extraction methods. These included either: phenol: chloroform extraction followed by alcohol precipitation (TRIzol), phenol:chloroform followed by solid-phase extraction (column-based; miRVana and miRNeasy) and solid-phase separation with/without affinity resin (Norgen total and Isolate II). We then evaluated each extraction method for the quality and quantity of RNA recovered, and the expression of miRNAs and target genes. RESULTS:We identified differences between each of the RNA extraction methods in the quantity and quality of RNA samples, and in the analysis of miRNA and target gene expression. For the purposes of consistency in quantity, quality and high recovery of miRNAs from tissues, we identified that Phenol:chloroform phase separation combined with silica column-based solid extraction method was preferable (miRVana microRNA isolation). We also identified a method that is not appropriate for miRNA analysis from tissue samples (Bioline Isolate II). For target gene expression any of the kits could be used to analyse mRNA, but if interested in analysing mRNA and miRNA from the same RNA samples some methods should be avoided. CONCLUSIONS:Different methods used to isolate miRNAs will yield different results and therefore a robust RNA isolation method is required for reproducibility. Researchers should optimise these methods for their specific application and keep in mind that "total RNA" extraction methods do not isolate all types of RNA equally.
Project description:The first step in biomarkers discovery is to identify the best protocols for their purification and analysis. This issue is critical when considering peripheral blood samples (plasma and serum) that are clinically interesting but meet several methodological problems, mainly complexity and low biomarker concentration. Analysis of small molecules, such as circulating microRNAs, should overcome these disadvantages. The present study describes an optimal RNA extraction method of microRNAs from human plasma samples. Different reagents and commercially available kits have been analyzed, identifying also the best pre-analytical conditions for plasma isolation. Between all of them, the column-based approaches were shown to be the most effective. In this context, miRNeasy Serum/Plasma Kit (from Qiagen) rendered more concentrated RNA, that was better suited for microarrays studies and did not require extra purification steps for sample concentration and purification than phenol based extraction methods. We also present evidences that the addition of low doses of an RNA carrier before starting the extraction process improves microRNA purification while an already published carrier dose can result in significant bias over microRNA profiles. Quality controls for best protocol selection were developed by spectrophotometry measurement of contaminants and microfluidics electrophoresis (Agilent 2100 Bioanalyzer) for RNA integrity. Selected donor and patient plasma samples and matched biopsies were tested by Affymetrix microarray technology to compare differentially expressed microRNAs. In summary, this study defines an optimized protocol for microRNA purification from human blood samples, increasing the performance of assays and shedding light over the best way to discover and use these biomarkers in clinical practice.
Project description:MicroRNAs (miRNAs) are small RNAs that bind to mRNA targets and regulate their translation. A functional study of miRNAs and exploration of their utility as disease markers require miRNA extraction from biological samples, which contain large amounts of interfering compounds for downstream RNA identification and quantification. The most common extraction methods employ silica columns or the TRIzol reagent but give out low recovery for small RNAs probably due to their short strand lengths. Herein, we fabricated the titanium dioxide nanofibers using electrospinning to facilitate miRNA extraction and developed the optimal buffer conditions to improve miRNA recovery from biological matrices of cell lysate and serum. We found that our TiO2 fibers could obtain a recovery of 18.0 ± 3.6% for miRNA fibers while carrying out the extraction in the more complex medium of cell lysate, much higher than the 0.02 ± 0.0001% recovery from the commercial kit. The much improved extraction of miRNAs from our fibers could be originated from the strong coordination between TiO2 and RNA's phosphate backbone. In addition, the binding, washing, and elution buffers judiciously developed in the present study can achieve selective extraction of small RNA shorter than 500 nucleotides in length. Our results demonstrate that TiO2 nanofibers can work as a valuable tool for extraction of miRNAs from biological samples with high recovery. Graphical abstract Schematic for extraction of small RNAs using TiO2 nanofibers.
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.