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© MiRXES 2016 MiRXES IDEAL™ miRNA Assays and Services
© MiRXES 2016
MiRXES IDEAL™
miRNA Assays and Services
© MiRXES 2016
Increased SensitivityOptimized RT-qPCR primers and reagents to drive efficient target amplification from limiting amounts (≥1pg) of input RNA sample.
Improved SpecificityNo universal primers. Every assay utilizes three miRNA specific primers to discriminate single nucleotide differences.
Speedy DetectionRNA to Ct in less than 2 hours for faster turnaround and improved throughput.
Reliable DataAssays optimized by MIRXES’ proprietary algorithm and wet-lab validated with synthetic miRNA templates and RNA from biological samples.
ConvenienceComplete kit to minimize set-up time. Compatible with all major qPCR instruments.
Unique RT Primer: Conformational restricted miRNA specific RT primer efficiently hybridizes
to mature but not precursor form of target miRNA.
Specific Real-Time PCR Primers: miRNA specific forward and reverse real-time PCR
primers confer further specificity and enable robust amplification of amplicon.
Tailored RT-qPCR Reagents: Optimized RT and qPCR master mixes enhance signal to
noise ratio.
Re-defining miRNA Quantification with Sensitivity, Specificity and Speed
Unique Features
Assay PrincipleKey Benefits
2 h
Figure 1. miRNA Assay Principle
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miRNA % AT MiRXES Vendor A Vendor B Vendor C
hsa-miR-27a-5p 45% 12.6 16.5 18.1 N/A
hsa-miR-500a-5p 52% 12.5 16.3 19.8 15.8
hsa-miR-30e-3p 55% 13.1 14.8 17.2 14.5
hsa-miR-215-5p 62% 12.8 16.0 19.5 15.6
hsa-miR-32-3p 73% 13.0 21.1 21.2 N/A
Ct Value (108 copies; lower is better)
Figure 3. Ct values of MiRXES assays and 3 leading vendors in detecting 5 miRNAs
Superior Sensitivity
The unique three primer design of the IDEAL™ miRNA qPCR Assays yields class leading sensitivity compared to other miRNA detection systems (Figure 2). These assays are shown to have more consistent performance over miRNAs with varying sequences especially those with high AT content (Figure 3).
Figure 2. qPCR amplification curves of MIRXES assay and leading vendor
Leading Vendor
MiRXES
Greater Reproducibility
MiRXES IDEAL™ miRNA qPCR assays yielded highly reproducible results in profiling more than 200 miRNAs from 30 cancer sera over a year in two independent laboratories (R2> 0.95). The ease of use enables even first time users to generate consistent technical and biological replicates (Figure 4).
Figure 4. Measurement reproducibility
Unparalleled SpecificityThe combination of miRNA specific RT primer and nested qPCR primer pairs enables assays to efficiently discriminate highly homologous miRNA family members with single nucleotide difference.
Figure 5. Assay Specificity Test. Relative detection when assays are challenged with mismatched let-7 family members
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© MiRXES 2016
Tailored Solutions For Different Needs
Premium Biomarker Discovery Solution
Absolute expression quantification of 600-1000 miRNAs using MiRXES’ advanced cDNA library preparation and validated assays in highly controlled and automated laboratory.
State-of-the-art data processing, normalization and multi-variant statistical analysis.
8-12 weeks turnaround
IDEAL™ miRNA Knowledge PanelsReady-to-use assay panels for miRNAs relevant to specific areas of research.
Cancer Panel 352 miRNAStem Cell Panel 176 miRNABiofluid Panel 176 miRNA
Knowledge Panels
Profiling ServicesKnowledge Panel Profiling Service sample to answer screening service with miRNA Knowledge Panels, including sample isolation and RNA quality control services.
2-4 weeks turnaround.
IDEAL™ miRNA qPCR Assays Complete kit comprising of individual miRNA RT-qPCR assays with optimized RT-qPCR master mixes.
Ready-to-ship human, mouse, rat and virus assays (please refer to our assay list).
Customized Assay Design Assay design and wet-lab validation for novel miRNAs and non-catalogued species.
2-4 weeks turnaround.
Customized miRNA Panels
MiRNAs of interest pre-formatted in 96 or 384-well PCR plates, with optimized miRNA RT-qPCR master mixes provided in proportion.
Catalogued Assays
Customized Assays
• From RNA to answer, including spike-inRNA controls and data analysis template
• In 96/384 well format, compatible with all major qPCR instruments
• Sufficient for 50 RT and 100 qPCR reactions
• Reverse transcription can be multiplexed
MiRNA Target Validation
One time customization charges with standard pricing for subsequent assay & panel orders
MiRNA Target Identification
Pro
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2-6 weeks turnaround
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MiRXES IDEAL™ miRNA qPCR Kit
MiRXES IDEAL™ Spike-in RNA KitMiRXES IDEAL™ Spike-in RNAs are uniquely designed small RNAs (~22 nt) with sequences distinct from endogenous miRNAs. These Spike-in RNAs have been extensively tested and are compatible with various isolation methods, including phenol/chloroform-based, phenol-free, membrane, bead and precipitation methods, provided the method retains the small RNA fraction. MiRXES recommends the use of Spike-In RNAs to monitor and normalize experimental variations in sample RNA isolation, reverse transcription and qPCR.
MiRXES IDEAL™ Spike-in RNA Kit includes Spike-In RNAs, the corresponding RT-qPCR primers and optimized master mixes.
MiRNA qPCR Kit Content
MiRNA specific RT primers
cDNA synthesis buffer
Reverse transcriptase
MiRNA specific qPCR assays
miRNA qPCR master mix
Spike-in RNA Kit Content
Spike-in RNA 1 & 2
Spike-in RNA RT primer pool
cDNA synthesis buffer
Reverse transcriptase
Spike-in RNA 1 qPCR assay
Spike-in RNA 2 qPCR assay
miRNA qPCR master mix
Catalogue No. Description
MX003-2 MiRXES IDEAL™ miRNA qPCR Kit (2-miRNA Kit)
MX003-4 MiRXES IDEAL™ miRNA qPCR Kit (4-miRNA Kit)
MX003-10 MiRXES IDEAL™ miRNA qPCR Kit (10-miRNA Kit)
MC001 MiRXES IDEAL™ Spike-in RNA Kit
IDEAL™ miRNA qPCR and Spike-in RNA Kit
Catalogue No. Description
MX0C1 Customized miRNA qPCR Kit
MX0C2 Subsequent purchase of customized miRNA qPCR kit
IDEAL™ miRNA Kit Customization Service
Purchasing InformationFor more information please visit www.mirxes.com
Each MiRXES assay is designed in silico using our proprietary thermodynamics based algorithms with 30 design parameters and extensively wet-lab validated using both synthetic miRNA templates and RNA from biological samples. Users are able to select combinations of miRNAs from our growing library of validated human, mouse, rat and viral miRNA assays. For novel miRNAs and other non-catalogued species, MiRXES offers customized assay design and validation services. All miRNA qPCR kits include proportionally paired miRNA assays and RT-qPCR reagents.
© MiRXES 2016
MiRNAs are key regulators of stem cell homeostasis and differentiation. MiRNA expression patterns differ in various types of stem cells and as stem cells differentiate from a pluripotent state to specific cell lineage.
The MiRXES IDEALTM Stem Cell miRNA Knowledge panel consists of 176 miRNAs which allows researchers a broad-ranging overview on a convenient array.
MiRXES IDEALTM miRNA Knowledge Panels
Using MiRXES’ patented miRNA RT-qPCR technology, the IDEALTM
Knowledge Panels can produce results from RNA samples in just 2 hours.
RNA to Results in 2 hours
Reliable DataAssays optimized by MIRXES’ proprietary algorithm and wet-lab validated with synthetic miRNA templates and RNA from biological samples.
Ready-to-Use KitAll reagents, including 2 spike-in RNAs and inter-plate PCR calibrators areprovided proportionally to maximize convenience and user experience.
Automated Analysis TemplateAn open Excel template is available for automated analysis. The templateincludes options for expression normalization, data visualization and dataintegrity.
MiRNAs are actively and selectively secreted by cells as a means of intercellular communication. These circulating miRNAs have shown extraordinary stability in biofluidsand hold great potential as novel non-invasive markers for disease diagnosis, prognosis and monitoring.
The MiRXES IDEALTM Biofluid miRNA Knowledge panel consists of 176 miRNAs selected from in-house research of more than 20,000 human serum, plasma and urine specimen inhealthy and pathological conditions.
MiRXES IDEALTM miRNA knowledge panels are ready-to-use assay panels for miRNAs relevant to specific areas of research. Each panel profiles 176 or 352 miRNAs selected through extensive survey of in-house research data and published literature. All miRNA assays have been extensively wet-lab validated using synthetic miRNA templates and human sample RNA.
Currently, MiRXES offers three knowledge panels for miRNA research in stem cells, biofluids and cancers. Each kit is formatted to include all necessary reagents for the RNA-to-Ct workflow. Unique spike-in RNAs and inter-plate calibrators are built in to monitor and normalize technical variations from RNA isolation to qPCR. These kits are compatible with all major real-time qPCR platforms and are available in various pack sizes.
Stem Cell miRNA Panel
Biofluid miRNA Panel
MiRNAs are intimately involved in every hallmark of cancer. Detecting miRNA dysregulations could provide insights into cancer pathogenesis, drug response and recurrence.
The MiRXES IDEALTM Cancer miRNA Knowledge panel consists of 352 miRNAs highly associated with various cancer types and regulate key onco- and tumor suppressor genes and pathways.
Cancer miRNA Panel
Figure 6. miRNA qPCR panel work flow 6
© MiRXES 2016
Human fetal MSCs (7th passage) were differentiated into bone marrow and adipose lineages for 6 days. Five biological replicates were collected and profiled with IDEAL™ Stem Cell miRNA knowledge panel (Figure 7). One hundred nanograms of total RNA from each sample was used for reverse transcription. Of the 176 miRNAs, 165 miRNAs were detected in adipose lineage differentiation and 164 miRNAs were detected in cells from bone marrow lineages.
For experiments with limited sample size, MiRXES recommends a cut off of Ct 33 for higher data confidence. For human fetal MSC differentiation study,142 of the 164 detected miRNAs had Ct ≤ 33
Differential miRNA expression pattern observed as human fetal MSCs differentiate into adipose and bone marrow lineage.
Most significantly regulated miRNAs can be identified through the examination of p-value and fold change. Fold change can be calculated by 2-ΔΔCt
. Candidate miRNAs with most significant p-values and fold changes can be visualized through Volcano plot (Figure 10).
MiRXES offers individual miRNA qPCR assays for further validation of identified miRNA candidate. For more information, please visit:http://www.mirxes.com/life-science-products/individual-assays/
Application Note - IDEAL™ Stem Cell miRNA Panel
Figure 7. Experimental Set Up
Figure 9. Unsupervised clustering shows global change in miRNA expression pattern
Figure 8. Boxplot showing Ct number of miRNA tested on Stem Cell Panel and cut off criterion
Figure 10. Volcano plot showing ΔΔCt and p-value of tested miRNAs
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© MiRXES 2016
Four distinct cell types (HDFa, BE2C, HCT116 and U251) were profiled with IDEAL™ Cancer miRNA Panel. One hundred nanograms of RNA from each sample was used for reverse transcription. About 300 miRNAs were detected for each cell line,with 244 common miRNAs detected with Ct ≤ 33 across all 4 cell lines (Figure 6, 7). Inter-plate calibrator normalization and biological normalization by global mean or reference miRNAs were performed prior to inter-sample miRNA ΔCt calculation. MiRNA expression comparison in HFDa and U251 cells are visualized through a scatter plot. Data points that fall outside of ±1 cycles regression lines indicate miRNAs differentially expressed in the 2 cells (Figure 8).
Application Note – IDEAL™ Cancer miRNA Panel
IDEAL™ miRNA Panel Profiling ServicesLarge scale miRNA profiling can be challenging. MiRXES offers a comprehensive profiling service in our dedicated and high throughput miRNA biomarker discovery laboratory. The profiling service starts with a consultation with our product and service specialist to understand the needs of specific projects. A modularized workflow is then customized to best satisfy the these research needs and project budget. Customers can submit either samples or isolated RNA for miRNA profiling using one of our miRNA assay products. Extensive quality control measures are put in-place to ensure maximum data accuracy and quality. Data analysis and study report will be provided at the end of the service with consultation and technical recommendations for follow up steps.
Contact us to begin your miRNA discovery today!
Application Note – IDEAL™ Biofluid miRNA PanelMiRXES’ Biofluid miRNA knowledge panel was compared to equivalent products from two leading vendors using a common pool of human serum RNA. For each product, RNA extracted from 200 µL of pooled human serum was used at the start of respective workflows.Numbers of detected miRNAs were determined base on manufacturer’s instructions (Ct ≤ 33 for IDEAL Biofluid Panel). Of the 3 biofluid miRNA products, IDEAL Biofluid Panel detected the highest number of miRNAs (Figure 9). Separately, IDEAL Biofluid Panel was used to illustrate distinct serum miRNA profiles in normal and gastric cancer patient (Figure 10).
Figure 9. Comparison of panels
Figure 6. Number of miRNAs detected in different cell lines
Figure 7. Boxplot showing Ct number of miRNAs detected using Cancer miRNA Panel
Figure 10. miRNAs in gastric cancer serum and their ΔΔCt compared to pooled human serum
Figure 8. A comparison of miRNA expressions in U251 and HDFa
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© MiRXES 2016
MiRXES IDEAL™ Knowledge Panels
Catalogue No. Description
MP001-2 MiRXES IDEAL™ Biofluid miRNA Panel (2-Sample Kit)
MP001-6 MiRXES IDEAL™ Biofluid miRNA Panel (6-Sample Kit)
MP001-12 MiRXES IDEAL™ Biofluid miRNA Panel (12-Sample Kit)
MP001-24 MiRXES IDEAL™ Biofluid miRNA Panel (24-Sample Kit)
MP002-2 MiRXES IDEAL™ Stem Cell miRNA Panel (2-Sample Kit)
MP002-6 MiRXES IDEAL™ Stem Cell miRNA Panel (6-Sample Kit)
MP002-12 MiRXES IDEAL™ Stem Cell miRNA Panel (12-Sample Kit)
MP002-24 MiRXES IDEAL™ Stem Cell miRNA Panel (24-Sample Kit)
MP003-2 MiRXES IDEAL™ Cancer miRNA Panel (2-Sample Kit)
MP003-6 MiRXES IDEAL™ Cancer miRNA Panel (6-Sample Kit)
MP003-12 MiRXES IDEAL™ Cancer miRNA Panel (12-Sample Kit)
MP003-24 MiRXES IDEAL™ Cancer miRNA Panel (24-Sample Kit)
Catalogue No. Description
MS001 MiRXES IDEAL™ Biofluid Panel Profiling Service (12 samples minimum)
MS002 MiRXES IDEAL™ Stem Cell Panel Profiling Service (12 samples minimum)
MS003 MiRXES IDEAL™ Cancer Panel Profiling Service (12 samples minimum)
MS004 Customized Panel Profiling Services (12 samples minimum)
MiRXES IDEAL™ Knowledge Panels Profiling Service
MiRXES IDEAL™ Panel Customization Service
Catalogue No. Description
MP0C1 Customize Panel for 96 or 384-well plate (50 plates minimum)
MP0C2 Customized Panel ongoing purchase (12 plates minimum)
Ordering InformationFor more information please visit www.mirxes.com
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© MiRXES 2016
Premium Biomarker Discovery Solution
Catalogue No. Description
MSL001Absolute expression quantification of 600-1000 miRNAs using MiRXES’ advanced cDNA library preparation and validated assays in highly controlled and automated laboratory.
MSL002 Bioinformatics analysis and biomarker selection
MSL003 Customization of biomarker assay kit (ISO13485)
Leveraging on the core qPCR technology and validated assay library, MiRXES’ diagnostic arm has worked with numerous clinical and commercial partners to identify and validate biomarkers in both biofluids and tissues for a variety of diseases. The experience has enabled us to streamline and optimize a highly controlled and automated workflow for high throughput quantification of 600-1000 human miRNAs from minute amount of clinical specimen. To date, more than 20,000 serum, plasma, urine and FFPE tissue specimen have been profiled using the workflow. MiRXES now offers this unique solution for projects that require greater data granularity and depth.
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Ordering InformationPlease contact us at [email protected]
Unique Features
I. Sample focus approach – this workflow employs a unique cDNA array approach, where hundreds of clinical sample cDNAs are loaded into the same 384-well PCR plate with intra-plate positive and negative samples for greater control and accuracy.
II. Absolute quantification – 6-log serial dilutions of synthetic miRNA templates are concurrently reverse transcribed and amplified with isolated sample RNA, enabling absolute quantification of miRNA target instead of relative Ct comparison.
III. Multi-layer control measures – six unique spike-in RNAs are added in high, medium and low concentration at distinct steps of the workflow to monitor and normalize isolation efficiency, RT-qPCR efficiency. Reference samples and RNAs are included as inter-study normalizers.
IV. Advanced cDNA library – our unique approach to cDNA pre-amplification allows efficient and unbiased library preparation, thus enables the workflow to quantify up to 1000 miRNAs from RNA isolated from minute quantity of clinical samples (200 µl of biofluid, 2-3 standard FFPE tissue slices).
V. State-of-art bioinformatics – the use of multi-layer controls enables efficient data processing and normalization. Normalized data are analysed using multiple statistical methods including Principle Component Analysis, Data Randomization Test, Forward Logistic Regression and Support Vector Machine to identify potential biomarkers.
VI. Diagnostic kit prototyping – upon validation of identified biomarker, diagnostic kit
development services are available to translate the findings from bench to bedside.
© MiRXES 201611
© MiRXES 2016
ContactE: [email protected] W: mirxes.comP: +65 6779 7340
HQ2 Tukang Innovation Grove ,JTC MedTech Hub, #08-01 Singapore 618305
R & D Center10 Biopolis Road, Chromos, #03-01 Singapore 138670
For Research Use Only. Not for use in diagnostic procedures.Trademarks of MIRXES Pte Ltd and its affiliated companies: MiRXES® and MiRXES IDEALTM
