qScript microRNA cDNA Synthesis Kit

Reverse transcribe small, single-stranded RNAs with superior sensitivity

Features & Benefits

 

  • Sensitive reverse transcription of small, single-stranded RNA
  • Labeled universal cDNA supports seamless qPCR analysis with PerfeCTa miR Assays or user-defined assay primer (e.g. piRNA, lncRNA) together with PerfeCTa Universal PCR Primer
  • Optimized for use with PerfeCTa SYBR Green SuperMix
  • For more information on Quantabio's miRNA System click here.

 

qScript microRNA cDNA Synthesis Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.

Description

The qScript microRNA cDNA Synthesis Kit is an optimized reagent system that reverse transcribes small single-stranded RNA into 5'-labeled cDNA using either total RNA or miRNA enriched samples.

Single-stranded RNA is first polyadenylated by poly(A) polymerase before reverse transcription into universal cDNA using high performance qScript RT with a proprietary adapter oligo(dT) primer.  The universal cDNA template enables simple and cost-effective microRNA profiling when used together with wet-lab validated PerfeCTa microRNA Assays, PerfeCTa Universal PCR Primer and PerfeCTa SYBR Green SuperMix.

This complete kit includes positive (human) control primer, SNORD44, that can be used to quantitate ubiquitously expressed small nucleolar RNA. In addition, the kit contains 20% extra poly(A) polymerase reaction buffer and microRNA cDNA Reaction Mix to perform (-) poly(A) polymerase and (-) reverse transciptase control reactions.

Performance Data

Quanta microRNA Technology


Quanta microRNA Technology

The qScript microRNA cDNA Synthesis Kit is suitable for use with total RNA or preparations pre-enriched for microRNAs.  A poly(A) tail is added to microRNAs followed by cDNA synthesis using an adapter primer and qScript RT in a single-tube reaction. The resulting cDNA is ready for qPCR amplification with a Universal PCR Primer and your choice of microRNA Assays. This unique single-tube system provides ease-of-use and efficient cDNA synthesis for quantification of microRNAs with a high degree of sensitivity and specificity. PerfeCTa™ SYBR Green SuperMixes ensure compatibility with any real-time thermal cycler.


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microRNA Detection Range


Detection of rare microRNAs

Quanta's microRNA profiling system provides linear detection and quantification of microRNAs across total-RNA input levels spanning six orders of magnitude. This means microRNAs will be detected when present even when tissue is scarce or the microRNAs are rare. 


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Quanta - let-7a


qScript; microRNA Quantification System Comparison to Competitor (hsa-let-7a)

microRNA cDNA synthesis was performed starting with log-fold dilutions of human brain total RNA (from 1.0 &micro;g to 10 pg) using the qScript&trade; microRNA cDNA Synthesis Kit (Quanta) or the miScript Reverse Transcription Kit (Qiagen). 1/100th of each microRNA cDNA reaction was used in a 20 &microL qPCR amplification reaction specific for hsa-let-7a using each manufacturers validated microRNA assay, qPCR mix and recommended protocol. Averaged plots for triplicate qPCR reactions for each input quantity are shown. Cycling conditions for the miScript Primer Assay with the miScript SYBR Green PCR Kit (Qiagen) consisted of 94&deg;C, 15 min followed by 40 cycles of 94&deg;C, 15s; 55&deg;C, 30s and 70&deg;C, 30s. Cycling conditions for the PerfeCTa&reg; microRNA Assay and PerfeCTa&reg; SYBR&reg; Green SuperMix&trade; (Quanta) consisted of 95&deg;C, 2 min followed by 40 cycles of 95&deg;C, 5s and 60&deg;C, 30s. 


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Quanta vs ABI


qScript microRNA Quantification System Comparison to ABI (hsa-miR-124)

MicroRNA cDNA synthesis was performed starting with log-fold dilutions of human brain total RNA (from 1.0 &micro;g to 100 pg) using the qScript&trade; MicroRNA cDNA Synthesis Kit (Quanta) or the TaqMan&reg; microRNA Reverse Transcription Kit (ABI). 1/100th of each microRNA cDNA reaction was used in a 20 &micro;L qPCR amplification reaction specific for hsa-miR-124 using each manufacturers validated microRNA assay, qPCR mix and recommended protocol. Averaged plots for triplicate qPCR reactions for each input quantity are shown. Cycling conditions for the TaqMan MicroRNA Assay with TaqMan Universal PCR Master Mix (ABI) consisted of 95&deg;C, 5 min followed by 40 cycles of 95&deg;C, 15s; 60&deg;C, 1 min. Cycling conditions for the PerfeCTa&reg; MicroRNA Assay with PerfeCTa SYBR&reg; Green SuperMix&trade; (Quanta) consisted of 95&deg;C, 2 min followed by 40 cycles of 95&deg;C, 5s and 60&deg;C, 30s.


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What Customers Say
A well-tested and affordable microRNA profiling platform that delivers reproducible and reliable results.
Prof Dr Yigal Pinto, CSO
ACS Biomarker

Details

  • Contents
    • Poly(A) Tailing Buffer (5X)
    • Poly(A) Polymerase
    • MicroRNA cDNA Reaction Mix
    • qScript Reverse Transcriptase
    • PerfeCTa Universal PCR Primer
    • PerfeCTa Human Positive Control Primer
    • Nuclease-Free Water
  • Storage & Handling
    The kit components are stable for up to one year when stored at or below -20°C.
  • Related Resources
    Product Manuals
    Product Flyers
    Publications
    Modulation of the Mesenchymal Stem Cell Secretome Using Computer-Controlled Bioreactors: Impact on Neuronal Cell Proliferation, Survival and Differentiation
    Fábio G. Teixeira, Scientific Reports - 2016
    Abstract
    In recent years it has been shown that the therapeutic benefits of human mesenchymal stem/stromal cells (hMSCs) in the Central Nervous System (CNS) are mainly attributed to their secretome. The implementation of computer-controlled suspension bioreactors has shown to be a viable route for the expansion of these cells to large numbers. As hMSCs actively respond to their culture environment, there is the hypothesis that one can modulate its secretome through their use. Herein, we present data indicating that the use of computer-controlled suspension bioreactors enhanced the neuroregulatory profile of hMSCs secretome. Indeed, higher levels of in vitro neuronal differentiation and NOTCH1 expression in human neural progenitor cells (hNPCs) were observed when these cells were incubated with the secretome of dynamically cultured hMSCs. A similar trend was also observed in the hippocampal dentate gyrus (DG) of rat brains where, upon injection, an enhanced neuronal and astrocytic survival and differentiation, was observed. Proteomic analysis also revealed that the dynamic culturing of hMSCs increased the secretion of several neuroregulatory molecules and miRNAs present in hMSCs secretome. In summary, the appropriate use of dynamic culture conditions can represent an important asset for the development of future neuro-regenerative strategies involving the use of hMSCs secretome.
    Circulating Extracellular Vesicles Contain miRNAs and are Released as Early Biomarkers for Cardiac Injury
    Janine C.Deddens, Journal of Cardiovascular Translational Research - 2016
    Abstract
    Plasma-circulating microRNAs have been implicated as novel early biomarkers for myocardial infarction (MI) due to their high specificity for cardiac injury. For swift clinical translation of this potential biomarker, it is important to understand their temporal and spatial characteristics upon MI. Therefore, we studied the temporal release, potential source, and transportation of circulating miRNAs in different models of ischemia reperfusion (I/R) injury. We demonstrated that extracellular vesicles are released from the ischemic myocardium upon I/R injury. Moreover, we provided evidence that cardiac and muscle-specific miRNAs are transported by extracellular vesicles and are rapidly detectable in plasma. Since these vesicles are enriched for the released miRNAs and their detection precedes traditional damage markers, they hold great potential as specific early biomarkers for MI.
    De-Regulation of MicroRNA and Gene Expression in Diabetic Foot Ulcers Lead to a Dampened Inflammatory Response, Inefficient DNA Repair and Inhibition of Cell Migration - viewcontent.cgi
    Horacio A . Ramirez, University of Miami - 2016
    Abstract
    Diabetic foot ulcers (DFUs) are a common and severe type of chronic wound of which molecular pathophysiology is poorly understood, resulting in slow development of new and efficacious treatments. The aim of this study was to identify cellular functions that contribute to pathophysiology of DFUs as compared to the acute wounds, by using genomic profiling. I postulated that de-regulation of DFU-specific genes and microRNAs (miRs) play an important role in the development of a chronic wound phenotype. I utilized a bedside to-bench approach in which DFU tissue samples from patients, non-neuropathic diabetic (DFS) and non-diabetic foot skin (NFS) biopsies were used to generate microarray expression profiles and miR PCR expression profiles.
    Lactic Acid Suppresses IL-33-Mediated Mast Cell Inflammatory Responses via Hypoxia-Inducible Factor-1 -Dependent miR-155 Suppression
    Abebayehu, D, The Journal of Immunology - 2016
    Abstract
    Lactic acid (LA) is present in tumors, asthma, and wound healing, environments with elevated IL-33 and mast cell infiltration. Although IL-33 is a potent mast cell activator, how LA affects IL-33–mediated mast cell function is unknown. To investigate this, mouse bone marrow–derived mast cells were cultured with or without LA and activated with IL-33. LA reduced IL-33–mediated cytokine and chemokine production. Using inhibitors for monocarboxylate transporters (MCT) or replacing LA with sodium lactate revealed that LA effects are MCT-1– and pH-dependent. LA selectively altered IL-33 signaling, suppressing TGF-β–activated kinase-1, JNK, ERK, and NF-κB phosphorylation, but not p38 phosphorylation. LA effects in other contexts have been linked to hypoxia-inducible factor (HIF)-1α, which was enhanced in bone marrow–derived mast cells treated with LA. Because HIF-1α has been shown to regulate the microRNA miR-155 in other systems, LA effects on miR-155-5p and miR-155-3p species were measured. In fact, LA selectively suppressed miR-155-5p in an HIF-1α–dependent manner. Moreover, overexpressing miR-155-5p, but not miR-155-3p, abolished LA effects on IL-33–induced cytokine production. These in vitro effects of reducing cytokines were consistent in vivo, because LA injected i.p. into C57BL/6 mice suppressed IL-33–induced plasma cytokine levels. Lastly, IL-33 effects on primary human mast cells were suppressed by LA in an MCT-dependent manner. Our data demonstrate that LA, present in inflammatory and malignant microenvironments, can alter mast cell behavior to suppress inflammation.
    High-throughput miRNA sequencing and identification of biomarkers for forensically relevant biological fluids
    Sarah Seashols-Williams, ELECTROPHORESIS - 2016
    Abstract
    microRNAs (miRNAs) are small non-coding RNAs that regulate cellular processes through modulation of proteins at the translational level. They tend to be highly stable as compared to other RNA species due to their small size and protection by protein and/or lipid matrices. Thus, it is likely that miRNAs, when fully evaluated, will make excellent candidates for body fluid identification. miRNA analysis of body fluids has been the subject of some recent interest in the forensic community. In this study, small RNAs were isolated from individual donations of eight forensically relevant biological fluids (blood, semen, vaginal fluid, menstrual blood, saliva, urine, feces, and perspiration) and subjected to next generation sequencing using the Illumina® Hi-Seq platform. Sequencing reads were aligned and annotated against miRbase release 21, resulting in a list of miRNAs and their relative expression levels for each sample analyzed. Body fluids with high bacterial loads (vaginal fluid, saliva, and feces) yielded relatively low annotated miRNA counts, likely due to oversaturation of small RNAs from the endogenous bacteria. Both body-fluid specific (miRs-200b, 1246, 320c, 10b-5p, 26b, and 891a) and potential normalization miRNAs (let-7g and i) were identified for further analysis as potential body fluid identification tools for each body fluid. This article is protected by copyright. All rights reserved
    Click here to see all Publications
    FAQs
    What RNA isolation kit or protocols are compatible with the qScript microRNA Quantification System?
    Any RNA isolation kit or protocol that retains or enriches small RNAs is compatible. RNA isolation kits that enrich mRNA and exclude small RNAs are not compatible. Please refer to the user manual of your RNA isolation kit to be sure that it retains and does not exclude small RNAs. We have had good success with Trizol prepared total RNA. Also with cell lines you can use a direct cell lysis reagent (RNAGEM from ZyGEM) eliminating the RNA preparation step. We do not recommend using RNA preparations that are enriched for small RNAs because they can introduce a bias between different sample preparations.
    The microRNA kit arrived thawed without dry ice, will the kit still work?
    We have tested the kit after 1 day and 3 days at room temperature and the results were identical to material stored at -20 C. The functionality of the kit components can be tested using dilutions of synthetic microRNA oligonucleotides. In addition, the Human Control Primer (included in the qScript microRNA cDNA Synthesis Kit) can be used as a template for cDNA synthesis followed by qPCR. Start with at least a 1/1000 dilution of the human control primer and then make 3 or more 1/10 dilutions. Use 5 uL of each dilution in 10 uL reactions with and without the poly(A) polymerase (substituting water for enzyme in the no-poly(A) polymerase reactions). Run the reverse transcriptase reactions for each and dilute the cDNAs to 100 uL by adding 80 uL of TE0.1 (10 mM Tris-HCl, pH 8, 0.1 mM EDTA) or water. Run 1 to 5 uL of the diluted cDNA reactions in 25 uL qPCR reactions using the human control assay primer and universal PCR primer. You should be able to generate a standard curve and see significant differences in Ct values between the samples prepared with and without poly(A) polymerase.
    Can the qScript microRNA system be used with serum and plasma samples?
    The qScript microRNA Quantification System can be used with serum and plasma samples. Quanta participated in the miRQC study where the microRNA kit performed very well in every category with each sample including the most challenging serum samples. Serum/plasma RNAs can be purified using TRIzol, TRIzol LS or Qiagen kits (either miRNeasy Mini or miRNeasy Serum/Plasma). Since the RNA concentrations are low in serum and plasma samples, it is important to include a co-precipitant like glycogen or MS2 phage RNA at the RNA precipitation step. There are numerous references on the topic in the literature (see whitepaper from Qiagen). We would recommend using a synthetic RNA oligo spike-in control. We offer 4 different C. elegans microRNA assays that can be used for this purpose. You must first order a custom RNA oligonucleotide synthesis for one of the C. elegans microRNAs. Samples are spiked with a dilution of the synthetic RNA oligo template immediately following lysis and homogenization of the sample. Add enough synthetic RNA oligo to each sample so that there will be at least 10,000 copies per well in each qPCR reaction. For example if 1/10 of the RNA prep was used to make cDNA and 1/100 of the cDNA was used in each qPCR then spike the sample with 107 copies of the synthetic RNA oligo. Use equal volumes of sample in the cDNA synthesis and qPCR reactions since RNA concentration cannot be accurately determined. The qPCR results of the C. elegans assay can then be used to normalize for the efficiency of the RNA sample preparation method. Since the typical small RNA controls are usually not detectable in serum and plasma samples you must select a panel of microRNA assays that are known to be detectable in serum. Following some preliminary experimentation, choose a subset of assays whose levels are determined to be invariant with respect to the biological model system of interest (for example normal and disease tissue) to use as data normalization controls.
    Can the qScript microRNA Quantification System be used to quantify mRNA as well as miRNA?
    Although the kit has been designed to work optimally for microRNA detection, mRNA will be represented in the microRNA cDNA population. The representation of mRNA, however, will favor amplicons designed near the 3’-end of transcripts since the reverse transcriptase reaction is primed using an oligo-dT adapter primer. Equal representation of the entire length of mRNA transcripts would require a random priming method.
    How does the qScript microRNA Quantification System distinguish mature microRNAs from precursor-microRNA (pre-miRNA) hairpin structures?
    The qScript microRNA Quantification System is specific for mature microRNAs. The PerfeCTa microRNA Assays primarily distinguish mature microRNAs from pre-microRNAs based on proprietary design features of the PerfeCTa microRNA assay primer. Efficacy of pre-miRNA as template also depends on how well the pre-miRNA can be poly(A) tailed. Recessed and blunt 3’-ends of pre-miRNA hairpins are a poor substrate for poly(A) polymerase tailing. In addition, the hairpin structure of pre-miRNAs may not be efficiently amplified during PCR due to the stability of the secondary structure of the template competing with the annealing of the PCR primer. Finally, if a pre-miRNA product is amplified it will typically be larger in size than the mature microRNA product and thus distinguished by melting at a higher temperature.
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