Quantification of Bf-miRNA
Bf-miRNA quantification research is still in its infancy. Nevertheless several good attempts were made at this end
Absolute and Direct MicroRNA Quantification Using DNA–Gold Nanoparticle Probes
This is very interesting and highly promising paper.
DNA–gold nanoparticle probes are implemented in a simple strategy for direct microRNA (miRNA) quantification. Fluorescently labeled DNA-probe strands are immobilized on PEGylated gold nanoparticles (AuNPs). In the presence of target miRNA, DNA–RNA heteroduplexes are formed and become substrate for the endonuclease DSN (duplex-specific nuclease). Enzymatic hydrolysis of the DNA strands yields a fluorescence signal due to diffusion of the fluorophores away from the gold surface. We show that the molecular design of our DNA–AuNP probes, with the DNA strands immobilized on top of the PEG-based passivation layer, results in nearly unaltered enzymatic activity toward immobilized heteroduplexes compared to substrates free in solution. The assay, developed in a real-time format, allows absolute quantification of as little as 0.2 fmol of miR-203. We also show the application of the assay for direct quantification of cancer-related miR-203 and miR-21 in samples of extracted total RNA from cell cultures. The possibility of direct and absolute quantification may significantly advance the use of microRNAs as biomarkers in the clinical praxis.
This is very interesting and highly promising paper.
DNA–gold nanoparticle probes are implemented in a simple strategy for direct microRNA (miRNA) quantification. Fluorescently labeled DNA-probe strands are immobilized on PEGylated gold nanoparticles (AuNPs). In the presence of target miRNA, DNA–RNA heteroduplexes are formed and become substrate for the endonuclease DSN (duplex-specific nuclease). Enzymatic hydrolysis of the DNA strands yields a fluorescence signal due to diffusion of the fluorophores away from the gold surface. We show that the molecular design of our DNA–AuNP probes, with the DNA strands immobilized on top of the PEG-based passivation layer, results in nearly unaltered enzymatic activity toward immobilized heteroduplexes compared to substrates free in solution. The assay, developed in a real-time format, allows absolute quantification of as little as 0.2 fmol of miR-203. We also show the application of the assay for direct quantification of cancer-related miR-203 and miR-21 in samples of extracted total RNA from cell cultures. The possibility of direct and absolute quantification may significantly advance the use of microRNAs as biomarkers in the clinical praxis.
Detection and quantification of extracellular microRNAs in murine biofluids
MicroRNAs (miRNAs) are short RNA molecules which regulate gene expression in eukaryotic cells, and are abundant and stable in biofluids such as blood serum and plasma. As such, there has been heightened interest in the utility of extracellular miRNAs as minimally invasive biomarkers for diagnosis and monitoring of a wide range of human pathologies. However, quantification of extracellular miRNAs is subject to a number of specific challenges, including the relatively low RNA content of biofluids, the possibility of contamination with serum proteins (including RNases and PCR inhibitors), hemolysis, platelet contamination/activation, a lack of well-established reference miRNAs and the biochemical properties of miRNAs themselves. Protocols for the detection and quantification of miRNAs in biofluids are therefore of high interest.
The following protocol was validated by quantifying miRNA abundance in C57 (wild-type) and dystrophin-deficient (mdx) mice. Important differences in miRNA abundance were observed depending on whether blood was taken from the jugular or tail vein. Furthermore, efficiency of miRNA recovery was reduced when sample volumes greater than 50 μl were used.
Here we describe robust and novel procedures to harvest murine serum/plasma, extract biofluid RNA, amplify specific miRNAs by RT-qPCR and analyze the resulting data, enabling the determination of relative and absolute miRNA abundance in extracellular biofluids with high accuracy, specificity and sensitivity.
Here we describe robust and novel procedures to harvest murine serum/plasma, extract biofluid RNA, amplify specific miRNAs by RT-qPCR and analyze the resulting data, enabling the determination of relative and absolute miRNA abundance in extracellular biofluids with high accuracy, specificity and sensitivity.
Absolute quantification of microRNAs by using a universal reference
MicroRNAs (miRNAs) are a species of small RNAs approximately 21-23-nucleotides long that have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous PCR-, sequencing-, or hybridization-based methods have been established to identify and quantify miRNAs. Their short length results in a high dynamic range of melting temperatures and therefore impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here, we present a microarray-based approach for global and absolute quantification of miRNAs. The method relies on the parallel hybridization of the sample of interest labeled with Cy5 and a universal reference of 954 synthetic miRNAs in equimolar concentrations that are labeled with Cy3 on a microarray slide containing probes for all human, mouse, rat, and viral miRNAs (miRBase 12.0). Each single miRNA is quantified with respect to the universal reference canceling biases related to sequence, labeling, or hybridization. We demonstrate the accuracy of the method by various spike-in experiments. Furthermore, we quantified miRNA copy numbers in liver samples and CD34(+)/CD133(-) hematopoietic progenitor cells
High-performance quantification of mature microRNAs by real-time RT-PCR using deoxyuridine-incorporated oligonucleotides and hemi-nested primers
MicroRNAs are small noncoding RNAs that serve as important regulators of eukaryotic gene expression and are emerging as novel diagnostic and therapeutic targets for human diseases. Robust and reliable detection of miRNAs is an essential step for understanding the functional significance of these small RNAs in both physiological and pathological processes. Existing methods for miRNA quantification rely on fluorescent probes for optimal specificity. In this study, we developed a high-performance real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay that allows specific and rapid detection of mature miRNAs using a fast thermocycling profile (10 sec per cycle). This assay exhibited a wide dynamic range (>7 logs) and was capable of detecting miRNAs from as little as 1 pg of the total RNA or as few as 10 cells. The use of modified reverse-transcription oligonucleotides with a secondary structure and hemi-nested reverse PCR primers allowed excellent discrimination of mature miRNAs from their precursors and highly homologous family members using SYBR Green I. Using a novel approach involving uracil-DNA glycosylase treatment, we showed that carryover of the reverse transcription oligonucleotide to the PCR can be successfully eliminated and discrimination between miRNA homologs could be further enhanced. These assays were further extended for multiplexed detection of miRNAs directly from cell lysates without laborious total RNA isolation. With the robust performance of these assays, we identified several miRNAs that were regulated by glial cell-line-derived neurotrophic factor in human glioblastoma cells. In summary, this method could provide a useful tool for rapid, robust, and cost-effective quantification of existing and novel miRNAs.
Development of Plasmid Calibrators for Absolute Quantification of miRNAs by Using Real-Time qPCR
MicroRNAs (miRNAs) are small noncoding RNAs of approximately 18 to 25 nucleotides in length that negatively regulate gene expression via either the degradation or translational inhibition of their target mRNAs. Because miRNAs are essential for the regulation of critical physiological processes as well as a variety of pathological events, they have emerged as a novel class of molecular diagnostic biomarkers and therapeutic agents or targets. Accordingly, the need for novel methods for the quantification of miRNA has increased due to interest in their clinical implications. Currently, real-time quantitative polymerase chain reaction (qPCR) is considered the most robust technology for nucleic acid quantification. Different tools for miRNA quantification by using qPCR are now commercially available, but only relative quantification strategies have been reported. This situation may be partly due to the difficulty in obtaining an appropriate molecule with which to establish an miRNA calibration range. Here, we describe a rapid and convenient strategy for the development of a calibrator, which enables the absolute quantification of miRNAs by using qPCR and allows the cloning of a synthetic sequence of interest instead of a PCR product into a plasmid.
Quantification of circulating miRNAs by droplet digital PCR: comparison of EvaGreen- and TaqMan-based chemistries
Droplet digital PCR (ddPCR) has been successfully used with TaqMan assays to assess gene expression through the quantification of mRNA and miRNA. Recently, a new ddPCR system that can also run DNA-binding dye-based assays has been developed but it has not yet been tested for miRNA. We tested and compared the feasibility of quantifying miRNA with the new QX200 Droplet Digital PCR system when used with EvaGreen dye- and TaqMan probe-based assays. RNA from plasma and serum of 28 patients with cancer and healthy persons was reverse-transcribed and quantified for two circulating miRNAs and one added exogenous miRNA, with both EvaGreen dye-based miRCURY LNA miRNA assays and TaqMan assays. Amplification and detection of target miRNAs were performed on the QX200 ddPCR system. Conditions required to run miRCURY LNA miRNA assays were optimized. The EvaGreen-based assay was precise, reproducible over a range of concentrations of four orders of magnitude, and sensitive, detecting a target miRNA at levels down to 1 copy/μL. When this assay was compared with TaqMan assays, high concordance was obtained for two endogenous miRNAs in serum and plasma (Pearson r > 0.90). EvaGreen dye-based and TaqMan probe-based assays can be equally used with the ddPCR system to quantify circulating miRNAs in human plasma and serum. This study establishes the basis for using EvaGreen dye-based assays on a ddPCR system for quantifying circulating miRNA biomarkers and potentially other low-abundance RNA biomarkers in human biofluids. See all the articles in this CEBP Focus section, "Biomarkers, Biospecimens, and New Technologies in Molecular Epidemiology.
Nanopore Single-Molecule Detection of Circulating MicroRNAs
MicroRNAs (miRNAs) are a class of tiny noncoding RNAs that play an important role in regulating every aspect of cellular activities. Dysfunctional expression of miRNAs disrupts normal biological processes, leading to the development of various diseases including cancer. Circulating miRNAs are being investigated as biomarkers with a potential for noninvasive disease detection. This demands the development of new technologies to accurately detect miRNAs with short assay time and affordable cost. We have proposed a nanopore single-molecule method for accurate, label-free detection of circulating miRNAs without amplification of the target miRNA. This concise protocol describes how to device a protein nanopore to quantify target miRNAs in RNA extraction, and discusses at the end the advantages, challenges, and broad impact of the nanopore approach for miRNA detection.
MicroRNAs (miRNAs) are a class of tiny noncoding RNAs that play an important role in regulating every aspect of cellular activities. Dysfunctional expression of miRNAs disrupts normal biological processes, leading to the development of various diseases including cancer. Circulating miRNAs are being investigated as biomarkers with a potential for noninvasive disease detection. This demands the development of new technologies to accurately detect miRNAs with short assay time and affordable cost. We have proposed a nanopore single-molecule method for accurate, label-free detection of circulating miRNAs without amplification of the target miRNA. This concise protocol describes how to device a protein nanopore to quantify target miRNAs in RNA extraction, and discusses at the end the advantages, challenges, and broad impact of the nanopore approach for miRNA detection.
Exponential Strand-Displacement Amplification for Detection of MicroRNAs
MicroRNAs (miRNAs) are promising targets for disease diagnosis. However, miRNA detection requires rapid, sensitive, and selective detection to be effective as a diagnostic tool. Herein, a miRNA-initiated exponential strand-displacement amplification (SDA) assay was reported. With the Klenow fragment, nicking enzyme Nt.AlwI, and two primers, the miRNA target can trigger two cycles of nicking, polymerization, and displacement reactions. These reaction cycles amplified the target miRNA exponentially and generated dsDNAs detectable with SYBR Green I in real-time PCR. As low as 16 zmol of the target miRNA was detected by this one-pot assay within 90 min, and the dynamic range spanned over 9 orders of magnitude. Negligible impact from the complex biological matrix was observed on the amplification reaction, indicating the assay’s capability to directly detect miRNAs in biofluids
MicroRNAs (miRNAs) are promising targets for disease diagnosis. However, miRNA detection requires rapid, sensitive, and selective detection to be effective as a diagnostic tool. Herein, a miRNA-initiated exponential strand-displacement amplification (SDA) assay was reported. With the Klenow fragment, nicking enzyme Nt.AlwI, and two primers, the miRNA target can trigger two cycles of nicking, polymerization, and displacement reactions. These reaction cycles amplified the target miRNA exponentially and generated dsDNAs detectable with SYBR Green I in real-time PCR. As low as 16 zmol of the target miRNA was detected by this one-pot assay within 90 min, and the dynamic range spanned over 9 orders of magnitude. Negligible impact from the complex biological matrix was observed on the amplification reaction, indicating the assay’s capability to directly detect miRNAs in biofluids
Designing a Polycationic Probe for Simultaneous Enrichment and Detection of MicroRNAs in a Nanopore
The nanopore sensor can detect cancer-derived nucleic acid biomarkers such as microRNAs (miRNAs), providing a noninvasive tool potentially useful in medical diagnostics. However, the nanopore-based detection of these biomarkers remains confounded by the presence of numerous other nucleic acid species found in biofluid extracts. Their nonspecific interactions with the nanopore inevitably contaminate the target signals, reducing the detection accuracy. Here we report a novel method that utilizes a polycationic peptide-PNA probe as the carrier for selective miRNA detection in the nucleic acid mixture. The cationic probe hybridized with microRNA forms a dipole complex, which can be captured by the pore using a voltage polarity that is opposite the polarity used to capture negatively charged nucleic acids. As a result, nontarget species are driven away from the pore opening, and the target miRNA can be detected accurately without interference. In addition, we demonstrate that the PNA probe enables accurate discrimination of miRNAs with single-nucleotide difference. This highly sensitive and selective nanodielectrophoresis approach can be applied to the detection of clinically relevant nucleic acid fragments in complex samples.
http://pubs.acs.org/doi/abs/10.1021/nn305789z
Simultaneous detection of multiple microRNAs for expression profiles of microRNAs in lung cancer cell lines by capillary electrophoresis with dual laser-induced fluorescence
MicroRNAs (miRNAs) are small, endogenous, single-stranded, noncoding RNAs. Circulating miRNAs are being considered as promising disease biomarkers. Indeed, single miRNAs have been associated with a wide variety of disease conditions and can target multiple mRNAs; therefore, several miRNAs may be simultaneously involved in disease progression and development. In this study, we developed a capillary electrophoresis with dual laser-induced fluorescence (CE with dual LIF) method using two color laser excitations for simultaneous determination of multiple miRNAs. Target miRNAs were hybridized with 6-FAM- or Cy5-labeled DNA probes for simultaneous determination of multiple miRNAs at excitation wavelengths of 488 and 635 nm. The hybridized miRNAs were separated using CE with dual LIF and detected within 13 min at excitation wavelengths of 488 and 635 nm without any interference or crosstalk. Additionally, the proposed approach was used successfully to detect and evaluate levels of several endogenous miRNAs from lung cancer cell lines. These results showed the potential of CE with dual LIF for fast, specific, simultaneous analysis of multiple miRNAs in cell extracts, biofluids, and tissues
http://www.sciencedirect.com/science/article/pii/S002196731301515X
MicroRNAs (miRNAs) are small, endogenous, single-stranded, noncoding RNAs. Circulating miRNAs are being considered as promising disease biomarkers. Indeed, single miRNAs have been associated with a wide variety of disease conditions and can target multiple mRNAs; therefore, several miRNAs may be simultaneously involved in disease progression and development. In this study, we developed a capillary electrophoresis with dual laser-induced fluorescence (CE with dual LIF) method using two color laser excitations for simultaneous determination of multiple miRNAs. Target miRNAs were hybridized with 6-FAM- or Cy5-labeled DNA probes for simultaneous determination of multiple miRNAs at excitation wavelengths of 488 and 635 nm. The hybridized miRNAs were separated using CE with dual LIF and detected within 13 min at excitation wavelengths of 488 and 635 nm without any interference or crosstalk. Additionally, the proposed approach was used successfully to detect and evaluate levels of several endogenous miRNAs from lung cancer cell lines. These results showed the potential of CE with dual LIF for fast, specific, simultaneous analysis of multiple miRNAs in cell extracts, biofluids, and tissues
http://www.sciencedirect.com/science/article/pii/S002196731301515X
Nanopore single-molecule dielectrophoretic detection of cancer-derived MicroRNA biomarkers
The nanopore-based single-molecule biosensor has been extensively investigated for various biomedical detections. It has demonstrated the potential in gene sequencing and diagnosis-oriented biomarker detection such as microRNAs. In real-time detection, however, samples extracted from bio-fluids contain various non-target nucleic acids components. These components can cause severely influence the target detection accuracy. We have discovered that a polycationic probe can solve this issue. The polycationic peptide domain of the probe can separate the target probe complex from free nucleic acids, and only lead the complex into the pore, therefore realizing simultaneous enrichment and detection of target microRNAs. This study establish a universal approach to detecting any short pathogenic nucleic acids fragment in complex samples
The nanopore-based single-molecule biosensor has been extensively investigated for various biomedical detections. It has demonstrated the potential in gene sequencing and diagnosis-oriented biomarker detection such as microRNAs. In real-time detection, however, samples extracted from bio-fluids contain various non-target nucleic acids components. These components can cause severely influence the target detection accuracy. We have discovered that a polycationic probe can solve this issue. The polycationic peptide domain of the probe can separate the target probe complex from free nucleic acids, and only lead the complex into the pore, therefore realizing simultaneous enrichment and detection of target microRNAs. This study establish a universal approach to detecting any short pathogenic nucleic acids fragment in complex samples
Quantification of Circulating miRNAs in Plasma : Effect of Preanalytical and Analytical Parameters on Their Isolation and Stability
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.
http://www.sciencedirect.com/science/article/pii/S1525157813001372
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.
http://www.sciencedirect.com/science/article/pii/S1525157813001372
Detection of miRNAs with a nanopore single-molecule counter
miRNAs are short noncoding RNA molecules that are important in regulating gene expression. Due to the correlation of their expression levels and various diseases, miRNAs are being investigated as potential biomarkers for molecular diagnostics. The fast-growing miRNA exploration demands rapid, accurate, low-cost miRNA detection technologies. This article will focus on two platforms of nanopore single-molecule approach that can quantitatively measure miRNA levels in samples from tissue and cancer patient plasma. Both nanopore methods are sensitive and specific, and do not need labeling, enzymatic reaction or amplification. In the next 5 years, the nanopore-based miRNA techniques will be improved and validated for noninvasive and early diagnosis of diseases.
http://informahealthcare.com/doi/abs/10.1586/erm.12.58
miRNAs are short noncoding RNA molecules that are important in regulating gene expression. Due to the correlation of their expression levels and various diseases, miRNAs are being investigated as potential biomarkers for molecular diagnostics. The fast-growing miRNA exploration demands rapid, accurate, low-cost miRNA detection technologies. This article will focus on two platforms of nanopore single-molecule approach that can quantitatively measure miRNA levels in samples from tissue and cancer patient plasma. Both nanopore methods are sensitive and specific, and do not need labeling, enzymatic reaction or amplification. In the next 5 years, the nanopore-based miRNA techniques will be improved and validated for noninvasive and early diagnosis of diseases.
http://informahealthcare.com/doi/abs/10.1586/erm.12.58
Determination of micro-RNA in cardiomyoblast cells using CE with LIF detection
Micro-RNAs (miRNAs) are small, endogenous, singlestranded, and noncoding RNAs. The miRNAs have been found to perform important functions in many cellular processes, such as development, proliferation, differentiation, and apoptosis. Circulating miRNAs have been proposed as emerging biomarkers in diseases such as cancer, diabetes, and cardiovascular disease including acute myocardial infarction (AMI). In this study, we developed CE with LIF (CE-LIF) using fluorescence-labeled DNA probe for determination of low abundance miRNA in cell extracts. The target miRNA is miRNA-499, a biomarker candidate of AMI with low abundance in biological samples. In order to measure the trace level of miRNA, we optimized the hybridization conditions such as hybridization time, temperature, and buffer solution. The highest fluorescence intensity of the hybridized miRNA-499 was found when hybridization was conducted at 40°C in 50 mM Tris-acetate (pH 8.0) buffer containing 50 mM NaCl, and 10 mM EDTA for 15 min. The hybridized miRNA-499 was detected in cultured H9c2 cardiomyoblast cells and the analysis of miRNA-499 was completed within 1 h using CE-LIF. These results showed the potential of CE for fast, specific, and sensitive high-throughput analysis of low-abundance miRNAs in cell extracts, biofluids, and tissues.
http://onlinelibrary.wiley.com/doi/10.1002/elps.201200442/abstract?userIsAuthenticated=false&deniedAccessCustomisedMessage=
Micro-RNAs (miRNAs) are small, endogenous, singlestranded, and noncoding RNAs. The miRNAs have been found to perform important functions in many cellular processes, such as development, proliferation, differentiation, and apoptosis. Circulating miRNAs have been proposed as emerging biomarkers in diseases such as cancer, diabetes, and cardiovascular disease including acute myocardial infarction (AMI). In this study, we developed CE with LIF (CE-LIF) using fluorescence-labeled DNA probe for determination of low abundance miRNA in cell extracts. The target miRNA is miRNA-499, a biomarker candidate of AMI with low abundance in biological samples. In order to measure the trace level of miRNA, we optimized the hybridization conditions such as hybridization time, temperature, and buffer solution. The highest fluorescence intensity of the hybridized miRNA-499 was found when hybridization was conducted at 40°C in 50 mM Tris-acetate (pH 8.0) buffer containing 50 mM NaCl, and 10 mM EDTA for 15 min. The hybridized miRNA-499 was detected in cultured H9c2 cardiomyoblast cells and the analysis of miRNA-499 was completed within 1 h using CE-LIF. These results showed the potential of CE for fast, specific, and sensitive high-throughput analysis of low-abundance miRNAs in cell extracts, biofluids, and tissues.
http://onlinelibrary.wiley.com/doi/10.1002/elps.201200442/abstract?userIsAuthenticated=false&deniedAccessCustomisedMessage=
Detection and quantification of extracellular microRNAs in murine biofluids
MicroRNAs (miRNAs) are short RNA molecules which regulate gene expression in eukaryotic cells, and are abundant and stable in biofluids such as blood serum and plasma. As such, there has been heightened interest in the utility of extracellular miRNAs as minimally invasive biomarkers for diagnosis and monitoring of a wide range of human pathologies. However, quantification of extracellular miRNAs is subject to a number of specific challenges, including the relatively low RNA content of biofluids, the possibility of contamination with serum proteins (including RNases and PCR inhibitors), hemolysis, platelet contamination/activation, a lack of well-established reference miRNAs and the biochemical properties of miRNAs themselves. Protocols for the detection and quantification of miRNAs in biofluids are therefore of high interest.
The following protocol was validated by quantifying miRNA abundance in C57 (wild-type) and dystrophin-deficient (mdx) mice. Important differences in miRNA abundance were observed depending on whether blood was taken from the jugular or tail vein. Furthermore, efficiency of miRNA recovery was reduced when sample volumes greater than 50 μl were used.
Here we describe robust and novel procedures to harvest murine serum/plasma, extract biofluid RNA, amplify specific miRNAs by RT-qPCR and analyze the resulting data, enabling the determination of relative and absolute miRNA abundance in extracellular biofluids with high accuracy, specificity and sensitivity.
http://link.springer.com/article/10.1186/1480-9222-16-5
MicroRNAs (miRNAs) are short RNA molecules which regulate gene expression in eukaryotic cells, and are abundant and stable in biofluids such as blood serum and plasma. As such, there has been heightened interest in the utility of extracellular miRNAs as minimally invasive biomarkers for diagnosis and monitoring of a wide range of human pathologies. However, quantification of extracellular miRNAs is subject to a number of specific challenges, including the relatively low RNA content of biofluids, the possibility of contamination with serum proteins (including RNases and PCR inhibitors), hemolysis, platelet contamination/activation, a lack of well-established reference miRNAs and the biochemical properties of miRNAs themselves. Protocols for the detection and quantification of miRNAs in biofluids are therefore of high interest.
The following protocol was validated by quantifying miRNA abundance in C57 (wild-type) and dystrophin-deficient (mdx) mice. Important differences in miRNA abundance were observed depending on whether blood was taken from the jugular or tail vein. Furthermore, efficiency of miRNA recovery was reduced when sample volumes greater than 50 μl were used.
Here we describe robust and novel procedures to harvest murine serum/plasma, extract biofluid RNA, amplify specific miRNAs by RT-qPCR and analyze the resulting data, enabling the determination of relative and absolute miRNA abundance in extracellular biofluids with high accuracy, specificity and sensitivity.
http://link.springer.com/article/10.1186/1480-9222-16-5
