miRNA Extraction Research Articles

Oligometastatic prostate cancer patients stratification: A molecular signature identified by liquid biopsy.

TPS400 Background: Oligometastatic prostate cancer (OPC) may represent the initial step of an unavoidable, rapid progression to a polymetastatic state, or the expression of a real oligometastatic phenotype related to a condition of stable disease for a long time. In the last scenario, stereotactic body radiation therapy (SBRT) can be considered as a potentially curative treatment option for hormone-naïve OPC. We propose a prospective, explorative trail with the aim of identifying by liquid biopsy a molecular signature (genes or differential expression of miRNA), and related clonal evolution, underlying metastatic prostate cancer progression after a course of SBRT. Methods: Study population will be 30 adult, hormone-naïve OPC undergoing SBRT. Table 1 summarizes gene and miRNA panel for molecular analysis. For each patient, 15 ml of peripheral blood will be collected before and at the end of SBRT, every 3 months for the first year, then every 6 months until disease progression, when another blood sample will be collected in case of instrumental evidence of failure (using PET-Choline or CT-scan plus bone scintigraphy). 7.5 ml of peripheral blood will be centrifuged to separate sera and 7.5 ml of peripheral blood will be centrifuged to separate plasma from the other blood components, respectively. Sera and plasma will be stored at -20°C and centralized to CREA Laboratory at our Institution, then immediately processed for cell free DNA (cfDNA) and miRNA extraction for NGS of target genes (by Illumina platforms MiSeq and MiniSeq) and dPCR analysis. Study duration will be 36 months. This protocol has been written and will be conducted in agreement with either the Declaration of Helsinki and subsequent amendments and ICH Harmonized Tripartite Guideline for Good Clinical Practice, and has received approval of local Ethics Committee. Panel for analysis. [Table: see text]

Exosomal small RNA sequencing uncovers the microRNA dose markers for power frequency electromagnetic field exposure

Purpose: The potential health risks caused by power frequency electromagnetic field (PFEMF) have led to increase public health concerns. However, the diagnosis and prognosis remain challenging in determination of exact dose of PFEMF exposure.Materials and methods: Mice were exposed to different magnetic doses of PFEMF for the following isolation of serum exosomes, microRNAs (miRNAs) extraction and small RNA sequencing. After small RNA sequencing, bioinformatic analysis, quantitative real-time PCR (qRT-PCR) validation and serum exosomal miRNA biomarkers were determined.Results: Significantly changed serum exosomal miRNA as biomarkers of 0.1, 0.5, 2.5 mT and common PFEMF exposure were confirmed. Gene ontology (GO) and Kyoto encyclopaedia of genes and genomes (KEGG) pathway analysis of the downstream target genes of the above-identified exosomal miRNA markers indicated that, exosomal miRNA markers were predicted to be involved in critical pathophysiological processes of neural system and cancer- or other disease-related signalling pathways.Conclusions: Aberrantly-expressed serum exosomal miRNAs, including miR-128-3p for 0.1 mT, miR-133a-3p for 0.5 mT, miR-142a-5p for 2.5 mT, miR-218-5p and miR-199a-3p for common PFEMF exposure, suggested a series of informative markers for not only identifying the exact dose of PFEMF exposure, also consolidating the base for future clinical intervention.

MiRNA extraction from cell-free biofluid using protein corona formed around carboxyl magnetic nanoparticles.

MicroRNA (miRNA) in urine has been considered as a potential biomarker for early-stage diagnosis of multiple diseases like urinary system cancer, kidney injury and diabetes, owing to their many demonstrated advantages including long-term stability and noninvasiveness. However, the traditional enrichment and extraction processes of miRNAs from urine are cumbersome and tedious due to the low concentration and multiple carriers of miRNAs. Herein, we present a novel method to collect low concentrations of miRNAs from dilute solutions such as urine and cell culture medium. 10-nm core sized magnetic nanoparticles with carboxylic acid coating can adsorb low-concentration proteins, and form protein corona which makes them easy to aggregate and precipitate for subsequent isolation. In urine and cell culture medium, these nanoparticles can aggregate with proteins, including miRNAs-associated protein Argonaute 2 and microvesicle-related proteins, to form precipitates, so that miRNAs can be easily extracted from pellets by small amount of lysis buffer for subsequent analysis such as real-time PCR. Our method provides a facile way to enrich miRNAs from biofluids without the need of ultracentrifugation and immunoprecipitations, bringing remarkable convenience to miRNAs-based biomarker research.

Comparison of the extraction and determination of serum exosome and miRNA in serum and the detection of miR-27a-3p in serum exosome of ALS patients.

Amyotrophic Lateral Sclerosis (ALS) is a muscle-bone degenerative disease, which lacks a specific index for diagnosis. In our previous studies, we found that exosomes mediated the interaction mechanism between muscle and bone at the cellular level, and myoblast exosomes can transfer miR-27a-3p to promote osteoblast mineralization. Therefore, we suppose that the expression of miR-27a-3p in the serum exosomes of ALS patients also changes. In this study, we used healthy human serum as a sample to find out the conditions and methods for extraction and detection. Then through comparison of the expression of miR-27a-3p in the serum exosomes of 10 ALS patients and healthy subjects, we found that in the ALS patients miR-27a-3p was down-regulated, and may be involved in the development of ALS, and therefore has potential as a reference for the diagnosis of ALS in the clinic.

MiRNA Quantification Method Using Quantitative Polymerase Chain Reaction in Conjunction with C q Method.

MicroRNAs are small noncoding RNAs that function to regulate gene expression. In general, miRNAs are posttranscriptional regulators that imperfectly bind to the 3'untranslated region (3'UTR) of target mRNAs bearing complementary sequences, and target more than half of all protein-coding genes in the human genome. The dysregulation of miRNA expression and activity has been linked with numerous diseases, including cancer, cardiovascular diseases, neurodegenerative disorders, and diabetes. To better understand the relationship between miRNAs and human disease, a variety of techniques have been used to measure and validate miRNA expression in many cells, tissues, body fluids, and organs. For many years, quantitative polymerase chain reaction (qPCR) has been the gold standard for measuring relative gene expression, and is now also widely used to assess miRNA abundance. In this chapter, we describe a quick protocol for miRNA extraction, reverse transcription, qPCR, and data analysis.

Feasibility of miRNA Extraction and Amplification From Chicken Caecal Content in Eimeria tenella INFECTION

Feasibility of miRNA Extraction and Amplification From Chicken Caecal Content in Eimeria tenella INFECTION

How to Explore the Function and Importance of MicroRNAs: MicroRNAs Expression Profile and Their Target/Pathway Prediction in Bovine Ovarian Cells.

Micro RNAs (miRNA) are integral components of genetic regulatory networks and act by binding to the transcripts of their corresponding target genes, leading to a decrease in protein production levels either by mRNA degradation or by translational repression. While the role of miRNAs is ubiquitous, they have a particular importance with regard to cell differentiation. The miRNA-target mRNA interaction has a significant impact on many signaling pathways and the cross-talk between them; playing a regulatory role in a variety of different physiological processes within the cells. Ovarian follicle development is a physiological process that is not fully understood with regard to miRNA regulation; there are many questions that remain with respect to the molecular regulation of this important process. Bovine follicular cells are a good experimental model for the investigation of these mechanisms, having direct implications on reproductive health in humans. This chapter describes how differentially expressed miRNAs are identified in the granulosa and theca cells of dominant and subordinate bovine ovarian follicles and the identification of their associated targets and pathways. This chapter systematically describes how the granulosa and theca cells are dissected from the ovarian follicles. Afterward, we present a detailed protocol for miRNA extraction, based on a combined TRI reagent/column clean-up method, and also miRNA expression profiling using both microarray and RT-qPCR. In addition, an outline is provided of the bioinformatic analysis which enables the prediction of miRNAs targets. Pathways associated with the differentially expressed miRNAs are also elucidated using DIANA-miRPath software.

190 Profile of MicroRNAs of Equine Amniotic Mesenchymal Cell-Derived Microvesicles in Different Time-Span of In Vitro Culture

Mesenchymal stromal cells (MSC) were found to secrete many factors with therapeutic relevance for their antioxidants, antiapoptotic, antifibrotic, angiogenic, immunomodulatory, and chemiotactic activities. The culture supernatant of MSC (in our case, of equine amniotic-derived cells) is defined secretome or conditioned medium (CM) and it is composed of soluble and insoluble factors secreted by cells. Soluble factors are represented by cytokines and growth factors, whereas microvesicles (MV), recently demonstrated to be an integral component of cell-to-cell communication during tissue regeneration, represent insoluble factors (Bruno et al. 2009 J. Am. Soc. Nephrol. 20, 1053-1067). Our previous data showed that equine amniotic-derived CM administered in vivo in equine spontaneous tendon lesion is able to regenerate the injured tissue. overlapping the results obtained by using in vivo the cells of origin in the same pathology (Lange-Consiglio et al. 2013 Stem Cells Devel. 22, 3015-3024). We also studied the amniotic-derived MV and found that they are involved in down-regulation of pro-inflammatory genes in in vitro LPS-stressed equine tendon and endometrial cells (Lange-Consiglio et al. 2016 Stem Cells Devel. 25, 610-621; Perrini et al. 2016 Stem Cell Res. Ther. 7, 169). Usually, protocols to produce CM and MV are different: CM is collected after 48 to 96 h of culturing cells without renewal of tissue culture medium, whereas MV are collected after culturing cells overnight. In future comparative studies of regenerative medicine aiming to understand the efficacy of CM and MV, understanding the quality of secretion of cells according to culturing time is crucial. The aim of the present study was to evaluate whether different times of culture can influence the micro-RNA (miRNA) cargo of equine amniotic-derived cells and their MV. Biological triplicates of a pool of amniotic-derived cells from 2 amniotic membranes and their MV, collected after overnight or after 4 days, were used. Following miRNA extraction and library preparation, deep sequencing was carried out on Illumina HisSEqn 2000 (Illumina Inc., San Diego, CA, USA). Mirdeep2 on Illumina high-quality trimmed sequences was used to detect known miRNAs and to support the individuation of novel miRNAs. The analysis identified 335 Equus caballus miRNA, which were quantified in both amniotic cells and MV. Hierarchical clustering of 83 miRNA that were observed in all 12 samples clearly discriminated amniotic cells and MV. The profile of miRNA in AMC and MV was similar between overnight and 4 days of culture. Further functional studies on the predicted target genes and pathways involved in the biological effect of equine amniotic secretome will be performed.

Extraction of microRNAs from biological matrices with titanium dioxide nanofibers.

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.

MiR-608 regulating the expression of ribonucleotide reductase M1 and cytidine deaminase is repressed through induced gemcitabine chemoresistance in pancreatic cancer cells.

Gemcitabine resistance is the main problem in pancreatic adenocarcinoma patients. Hence, we aimed to identify the correlation between expression of RRM1 and CDA as the resistance genes and their predicted targeting miR-608 in the resistant pancreatic cancer cell lines to gemcitabine. Dual luciferase assay was performed to determine whether both RRM1 and CDA are targeted by miR-608 in 293T and pancreatic cancer cell lines. AsPC-1 and MIA PaCa-2 cell lines became gradually resistant to gemcitabine by exposing to the increasing doses of gemcitabine. After RNA and miRNAs extraction and cDNA conversion, the expressions of RRM1, CDA and miR-608 in all cell lines were studied by quantitative PCR. Pre-miR-608 transfection to the cell lines was done by calcium phosphate method. MTT assay was performed for analyzing the chemo sensitivity of different cell lines to gemcitabine. Luciferase assays showed that miR-608 targeted RRM1 and CDA genes in 293T, AsPC-1 and MIA PaCa-2 cell lines. Compared to parental cell line, resistant MIA PaCa-2 and AsPC-1 cells demonstrated increased expression of RRM1 and CDA. On the other hand the expression of miR-608 in resistant MIA PaCa-2 and AsPC-1 cells was lower than parental cells. Furthermore, transfection of MIA PaCa-2 and AsPC-1 cells by miR-608 lead to decreased expression of RRM1 and CDA and lowered viability of the cells in comparison with scrambled microRNA transfected cells. During resistance induction in pancreatic cancer cells, miR-608 which is targeting RRM1 and CDA is downregulated which leads to upregulation of these genes.

An improvement of miRNA extraction efficiency in human plasma.

MicroRNAs (miRNAs) are short noncoding RNA molecules that control the expression of mRNAs associated with various biological processes. Therefore, deregulated miRNAs play important roles in the pathogenesis of diseases. Numerous studies are aimed at discovering biomarkers of diseases or determining miRNA functions by monitoring circulating miRNAs in various biological sources such as plasma and urine. However, the analysis of miRNA in such fluids presents problems related to accuracy and reproducibility because of their low levels in biological fluids. Therefore, better extraction kits and more sensitive detection systems have been developed for improved and reproducible analysis of circulating miRNAs. However, new extraction methods are also needed to improve the yield of miRNAs for their reliable analysis from biological fluids. The combination of yeast transfer RNA (tRNA) and glycogen as carrier molecules and incubation durations were optimized to maximize extraction efficiency. The extraction recovery using a combination of yeast tRNA and glycogen was approximately threefold more than that by using glycogen or yeast tRNA alone. In addition, reproducible and accurate analysis of miRNAs can be carried out after extraction using a combination of yeast tRNA and glycogen without an impact on plasma components. Graphical abstract Steps of miRNA extraction in plasma.

Extraction and qPCR-Based Detection of miRNAs from Cultured PBMCs of Bubaline Origin.

MicroRNAs are small noncoding but functionally important RNA molecules that are involved in regulating diverse cellular, metabolic, and immune processes. Their small size necessitates modification in traditional acid phenol-chloroform based RNA isolation procedures to get highly enriched fraction of small RNA that includes miRNAs and siRNAs . Further, of the different methods available, real-time PCR is a powerful tool for precise and specific detection and quantification of miRNA. Moreover, real-time PCR is used to validate the screening or expression of miRNAs that are discovered during high-throughput sequencing, or microarray analysis. We demonstrate here the method of extraction of miRNAs from cultured PBMCs of bubaline origin followed by the qPCR-based (both SYBR green and TaqMan -based chemistries) identification of miRNAs expressed in response to TLR ligand stimulation.

Extraction and Analysis of Extracellular Vesicle-Associated miRNAs Following Antibody-Based Extracellular Vesicle Capture from Plasma Samples.

Extracellular vesicle (EV)-associated RNAs (EV-RNA) are under intense investigation due to their potential role in health and disease. Several approaches are currently employed to isolate blood-derived EVs for RNA analysis, most of which are either time-consuming and expensive, such as methods based on EVs physical properties (ultracentrifugation and Optiprep density gradient), or also copurify blood contaminants, mostly protein aggregates and immune complexes, (such as chemical precipitation). In addition, there is a lack of standardized protocols for the extraction of EV-RNA and very little consensus on the technological platforms and normalization tools for assessing the expression levels of different RNA species. These methodological issues complicate the comparison between independent data sets, potentially biasing results and conclusions.In this book chapter we propose a protocol that might overcome some of the abovementioned issues through antibody-based isolation of blood-derived EVs followed by extraction and expression analysis of small-RNA species (miRNA) by reverse transcriptase quantitative PCR (RT-qPCR). The advantages of immunoaffinity approaches over other isolation methods are multiple and include: (1) the selective enrichment of specific EV subpopulations with restricted tissue/cell origin, (2) reduction of matrix effects and blood contaminants that may confound miRNA profiling from complex biological fluids and (3) easy coupling to conventional quantitative assays (e.g., RT-qPCR). In conclusion, we describe a protocol for standard enrichment and quantitative analysis of EV-miRNAs from blood and we warrant for technological improvements, such as the use of novel biomaterials, surface chemistries, binding agents and assay/sensor design that may further improve it.

Isolation and Identification of miRNAs in exosomes derived from serum of colon cancer patients.

Objective: To isolate exosomes from the serum of colon cancer patients and identify RNAs in the small vesicles.Methods: ExoQuick-TC™ Exosome Precipitation Solution was used for exosome isolation and the shapes of exosomes were observed under a transmission electron microscope. Mass spectrometry was used to identify the classification of miRNAs encapsulated in exosomes, and the expression levels of miRNA-21,-133a, and -181b in exosomes were detected by RT-PCR.Results: Exosomes isolated from serum of colon cancer patients were circular-or oval-shaped and vary in size with a diameter of 40-100 nm. Mass spectrometry shows that the main RNAs in exosomes are small RNAs; the levels of these small RNAs in exosomes are significantly higher compared with fresh serum. There is still a tiny amount of small RNAs in exosome-free serum, but the amounts are significantly lower than that in exosomes. No more RNAs were detected in the repeated freezing and thawing serum, but there were still some RNAs detectable in the exosomes extracted from these serums. MiRNA-21, -133a and -181b can be detected in exosomes, and the level of miRNA-21 is associated with early diagnosis of colon cancer.Conclusion: This study proves that commercial kits for exosome separation are more convenient and time-saving and that mass spectrometry is capable of identifying the miRNAs in exosomes. Compared with direct extraction of miRNAs from the serum, the method of isolating exosomes from the serum firstly and then extracting miRNAs from the exosomes can enhance the stability and integrity of their inner miRNAs. Also, we demonstrate that the exosomal miRNA-21 expression is associated with the early diagnosis of colon cancer.

Towards Stratification of Lymphomas with Involvement of the Central Nervous System Using miRNA-Seq from Small Volumes of Cerebrospinal Fluid

Early diagnosis and therapy monitoring have improved overall survival of lymphoma patients. Biomarkers from body fluids are an active field of research for diagnosing and monitoring malignant diseases. MicroRNAs (miRNAs) are present in all body fluids and have already been described as biomarkers in lymphoma patients. However, only few studies addressed the impact of miRNAs as diagnostic tools for central nervous system (CNS) involvement of systemic lymphomas (CNS positive lymphomas). In addition, due to technical limitations such as low cerebrospinal fluid (CSF) starting amounts, translation into clinical application has been very limited.In order to facilitate development of an applicable diagnostic assay of circulating miRNAs from low CSF starting volumes (400ml), we compared quantitative realtime PCR (qRT-PCR) arrays with next generation sequencing (miRNA-Seq) and established a protocol to robustly quantify a miRNA signature for CNS positive lymphomas from diagnostic volumes of CSF. First, we could show that circulating miRNAs are highly stable in CSF and that miRNA levels can be assessed robustly with qRT-PCR and miRNA-Seq. For this purpose, miRNAs were extracted from 400µL of CSF using an optimized miRNA extraction protocol. MiRNAs were quantified by qRT-PCR with the Exiqon Human miRNome Panel v2 which assays 752 miRNAs or by miRNA-Seq of libraries prepared with the Seqmatic TailorMix v2 miRNA Sample Preparation Kit using the Illumina HiSeq.In total, eight patients with diffuse large B-cell lymphoma (DLBCL) that showed involvement of the central nervous system (CNS) were analyzed. Of each patient, two paired CSF samples were available both from initial diagnosis and complete remission after treatment. The presence of CNS manifestation was established via magnetic resonance imaging (MRI) and flow cytometry at the Erasmus MC Daniel den Hoed Cancer Center in Rotterdam. CSF from ten patients with cephalalgia was used as a control.After inter-plate calibration and normalization, 260 miRNAs were detected on average with the Exiqon Human miRNome Panel v2. In order to assess the probability of deriving false positive signals from e.g. primer dimers, two miRNome Panels (four 384-well plates) were processed without template. Surprisingly, after 40 cycles of qRT-PCR, 10% of assays showed amplicons in duplicates. Furthermore, false positive amplicons were not reproducible using this method, underlining that qRT-PCR had a low overall specificity that could not be adjusted by exclusion of erroneous amplicons.In contrast, miRNA seq produced a median of ~40,000 mappable reads per sample (range 2x104 - 1x106) with a Phred score of >20. Prediction analysis using all detectable miRNAs segregated CSF samples from CSF positive lymphoma patients before treatment and after treatment based on a 14-miRNA signature with a misclassification error of <0.1. Unsupervised clustering of the 14-miRNA signature correctly stratified 7/8 samples after treatment i.e. CSF negative lymphomas, while 4/5 samples from patients with CSF positive lymphomas clustered separately. Thus, using the expression levels of only 14 miRNAs determined with miRNA-Seq, the two disease stages could be differentiated with each group containing one misclassification.The robust quantification of miRNAs from small volumes of CSF with next generation sequencing has potential to monitor minimal residual disease diagnosis or even to stratify other pathologic entities such as dementias or inflammatory diseases where only unreliable markers or no biomarkers at all are currently available. DisclosuresMulaw:NuGEN: Honoraria.

Extraction of miRNAs from Formalin-Fixed Paraffin-Embedded (FFPE) Tissues.

miRNAs are small non-coding RNAs that regulate gene expression and are involved in numerous diseases ranging from osteoporosis, cardiovascular disease, and numerous cancer indications. Most of the clinical material that is accessible for study is archival FFPE blocks. Due to the harsh nature of fixation and embedding procedures involved in preserving clinical material, these samples are heavily fragmented and chemically cross-linked by formalin. miRNAs, due to their small size and increased stability, are easier to retrieve and study in these precious tissues. Therefore, miRNAs have become useful tools in the diagnosis, prognosis, and prediction of diseases. Due to their increased importance, isolation of miRNAs from FFPE material is extremely clinically relevant. Here, we describe the best method for miRNA extraction from FFPE tissue blocks and determine the Qiagen miRNeasy FFPE kit to be the best kit for this task.

Automation of DNA and miRNA co-extraction for miRNA-based identification of human body fluids and tissues.

In the last years, microRNA (miRNA) analysis came into focus in the field of forensic genetics. Yet, no standardized and recommendable protocols for co-isolation of miRNA and DNA from forensic relevant samples have been developed so far. Hence, this study evaluated the performance of an automated Maxwell® 16 System-based strategy (Promega) for co-extraction of DNA and miRNA from forensically relevant (blood and saliva) samples compared to (semi-)manual extraction methods. Three procedures were compared on the basis of recovered quantity of DNA and miRNA (as determined by real-time PCR and Bioanalyzer), miRNA profiling (shown by Cq values and extraction efficiency), STR profiles, duration, contamination risk and handling. All in all, the results highlight that the automated co-extraction procedure yielded the highest miRNA and DNA amounts from saliva and blood samples compared to both (semi-)manual protocols. Also, for aged and genuine samples of forensically relevant traces the miRNA and DNA yields were sufficient for subsequent downstream analysis. Furthermore, the strategy allows miRNA extraction only in cases where it is relevant to obtain additional information about the sample type. Besides, this system enables flexible sample throughput and labor-saving sample processing with reduced risk of cross-contamination.

Human serum miR-34a as an indicator of exposure to ionizing radiation.

Radiation exposure in industrial accidents or nuclear device attacks is a major public health concern. There is an urgent need for markers that rapidly identify people exposed to ionizing radiation (IR). Finding a blood-based marker is advantageous because of the ease of sample collection. This study was designed to test the hypothesis that serum miR-34a could serve as an indicator of exposure to IR. Therefore, 44 women with breast cancer, where radiotherapy was part of their therapeutic protocol, were investigated in this study. After demonstrating the appropriateness of our microRNA (miRNA) extraction efficiency and miRNA assay in human serum, we analyzed the miR-34a level in paired serum samples before and after radiotherapy. Fifty Gy X-ray irradiation in daily dose fractions of 2Gy, 5days per week, was used in this study. We demonstrated that IR significantly increased serum level of miR-34a. By measuring miR-34a in serum, we could distinguish irradiated patients with sensitivity of 65% and specificity of 75%. According to this study, serum miR-34a has the potential to be used as an indicator of radiation exposure.

Identification of extracellular miRNA in archived serum samples by next-generation sequencing from RNA extracted using multiple methods.

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s11033-016-4043-6) contains supplementary material, which is available to authorized users.

The choice of endogenous controls in exosomal microRNA assessments from biofluids.

The assessment of differentially expressed microRNAs in patients and healthy controls is important to identify potential tumor biomarkers. Recently, it has been shown that the microRNA levels in exosomes are more correlated with the clinical-pathological variables than vesicle-free microRNAs (miRNAs) in biofluids; therefore, there is an increasing interest in these specific evaluations. However, these measurements can be affected by experimental problems that not always are evaluated and/or by inadequate procedural choices. In particular, exosome isolation and miRNA extraction procedures are crucial to avoid contaminations, and even the choice of the most suitable purity controls is important. Moreover, a stable endogenous RNA should be used for normalization of miRNA expression obtained by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) in order to make these measures comparable among different samples. A rushed choice of the endogenous control can bias study conclusions without revealing inconsistencies. Unfortunately, a few studies systematically identified the best normalizer for their specific experimental context. Instead, sometimes, the normalization procedures were performed in a disputable way or the normalizer choices simply based on the previous literature. Here, we reviewed the studies where the exosomal miRNA profiling was assessed in human biofluids to point out the adopted procedures and the specific endogenous controls chosen for normalization.