EPIK miRNA Panel and Individuals Assays Better by Design - - PowerPoint PPT Presentation

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EPIK™ miRNA Panel and Individuals Assays

Better by Design

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Introduction

What are miRNAs

A microRNA (miRNA) is a small non-coding RNA molecule (containing ~22 nucleotides) which functions in RNA post-transcriptional regulation of gene expression.

Functions of miRNAs

MicroRNAs have been shown to be involved in a wide range of biological, developmental and physiological processes such as:

• Cell cycle control
• Stem cell differentiation
• Hematopoiesis
• Hypoxia
• Cardiac and skeletal muscle

development

• Neurogenesis
• Insulin secretion
• Cholesterol metabolism
• Aging
• Immune responses
• Viral replication
• Embryogenesis

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Why use qPCR

qPCR Microarray Sequencing Throughput time ~6 hours ~2 days 1–2 weeks Total RNA required 500 ng 100-1,000 ng 500-5,000 ng Dynamic range detected Six orders of magnitude Four orders of magnitude Five or more

• rders of

magnitude Infrastructure and technical requirements Few Moderate Substantial

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SYBR Based qPCR miRNA

The most commonly used universal tailing method, reduces specificity1 Some suppliers using modified RNA nucleotides (LNA) to increase hybridization properties, however, they can display poorer amplification efficiencies, reducing sensitivity2

• 1. Chugh, P. & D.P. Dittmer. Potential pitfalls in microRNA profiling. WIREs RNA 3:601-616 (2012).
• 2. Levin, J. Fiala, D. Samala, M. Kahn, J. & Peterson, R. Position-dependent effects of locked nucleic acid (LNA) on DNA sequencing and PCR primers. Nucleic

Acids Res. 34:e142 (2006).

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The use of miRNA-specific forward and reverse primers

• ffers an advantage in terms of

the specificity of amplification However TaqMan probes need to be designed for each miRNA and so is much more expensive

Probe Based qPCR miRNA

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New Probe Based qPCR miRNA

The addition of adaptors on both ends has an advantage in terms of the specificity as the probe has more space, additionally an amplification step increases sensitivity However the TaqMan probes still need to be designed for each miRNA and so is much more expensive. The extra steps also makes the whole process more time consuming and more prone to mistakes

Mature microRNA

Poly A Tailing Ligation Universal RT miR-Amp qPCR

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EPIK qPCR miRNA

The use a set of three miRNA- specific primers offers an advantage in terms of the specificity of amplification, compared with other assays of similar design Use of less expensive SYBR dye

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Feature comparison

Bioline Supplier T Supplier Q Supplier E Original Advanced

Speed

2hrs 3hrs - 6hrs 3.5hrs 4hrs 3hrs - 2 days

Sensitivity

1pg 100pg 1pg 10pg 10pg

Input RNA amount recommended for RT reaction

100ng 500ng 10ng 1mg 100ng

Specificity

Single base Single base Single base Single base Single base

Dynamic range

7 log 6-7 log 6 log 6-10 log 5 log

Volume of RNA/total volume RT reaction

6ml for RNA in a 20ml reaction 5ml for RNA in a 15ml reaction 2ml for RNA in a 5ml reaction 15ml for RNA in a 20ml reaction 2ml for RNA in a 10ml reaction

Components supplied

In 1 kit As 3 Kits As 3 Kits As 3 Kits As 3 Kits

Company confidential

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Sensitivity

Ct value (108 copies of templates)

miRNA % AT EPIK miRNA Supplier E Supplier T Supplier Q has-miR-27a-5p 45% 12.6 16.5 18.1 N/A has-miR-500a-5p 52% 12.5 16.3 19.8 15.8 has-miR-30e-3p 55% 13.1 14.8 17.2 14.5 has-miR-215-5p 62% 12.8 16.0 19.5 15.6 has-miR-232-3p 73% 13.0 21.1 21.2 N/A

108 copies 100 copies NTC

y = -3,332x + 39,348 R² = 0,9997

10 20 30 40 5 10

Ct Log (copies of template)

Assay Efficiency: 99.0%

The results illustrate the sensitivity (108 to 100 copies) and efficiency (99.97%) of the assay, allowing the detection of miRNAs at varying expression levels, including low expressers. Comparison of five miRNAs with increasing AT concentrations

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Sensitivity

Ensuring that 108 copies is always between 12.5 and 13 Ct means that all miRNAs whether they are expressed at high concentrations or low concentrations, ideal for analysis of multiple miRNA targets from a single sample or low-level RNA samples such as serum and plasma.

EPIK Supplier E

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Specificity

Relative detection miRNA real-time RT-PCR assay

(%) let-7a let-7b let-7c let-7d let-7e let-7f let-7g let-71 miR-98

U G A G G U A G U A G G U U G U A U A G U U let-7a

100% 0.0% 0.10% 1.20% 0.0% 0.50% 0.0% 0.0% 0.0%

U G A G G U A G U A G G U U G U G U G G U U let-7b

0.0% 100% 2.20% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0%

U G A G G U A G U A G G U U G U A U G G U U let-7c

0.80% 1.60% 100% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0%

U G A G G U A G U A A G U U G U A U C G U U let-7d

0.40% 0.0% 0.10% 100% 0.0% 0.0% 0.0% 0.0% 0.0%

U G A G G U A G U A G A U U G U A U A G U U let-7e

0.10% 0.0% 0.0% 0.0% 100% 0.0% 0.0% 0.0% 0.0%

U G A G G U A G U A G U U U G U A C A G U U

let-7f 0.10% 0.0% 0.0% 0.0% 0.0% 100% 0.0% 0.0% 0.0%

U G A G G U A G U A G U U U G U G C U G U U let-7g

0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 100% 0.0% 0.0%

A G A G G U A G U A G G U U G C A U A G U U let-71

0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 100% 0.0%

U G A G G U A G G A G G U U G U A U A G U U miR-98

0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 100%

Combination of miRNA specific RT primer and nested real-time PCR primer pairs enables EPIK miRNA assays to efficiently discriminate highly homologous miRNA family members with single nucleotide difference, giving virtually no cross-reactivity and so greater confidence in profiling results.

Relative detection across the Let-7 miRNA family shows at least 50-fold specificity and virtually no cross-reaction.

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Specificity comparison

Company confidential

EPIK miRNA real-time RT-PCR assay let-7a let-7b let-7c let-7d let-7e let-7f let-7g let-71 let-7a 100% 0.0% 0.10% 1.2% 0.0% 0.5% 0.0% 0.0% let-7b 0.0% 100% 2.2% 0.0% 0.0% 0.0% 0.0% 0.0% let-7c 0.8% 1.6% 100% 0.0% 0.0% 0.0% 0.0% 0.0% let-7d 0.4% 0.0% 0.1% 100% 0.0% 0.0% 0.0% 0.0% let-7e 0.1% 0.0% 0.0% 0.0% 100% 0.0% 0.0% 0.0% let-7f 0.1% 0.0% 0.0% 0.0% 0.0% 100% 0.0% 0.0% let-7g 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 100% 0.0% let-71 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 100% miRNA real-time RT-PCR assay let-7a let-7b let-7c let-7d let-7e let-7f let-7g let-71 let-7a 100% 0.0% 0.0% 0.0% 4.0% 2.0% 0.0% 0.0% let-7b 0.0% 100% 3.0% 0.0% 0.0% 0.0% 0.0% 0.0% let-7c 1.0% 2.0% 100% 0.0% 0.0% 0.0% 0.0% 0.0% let-7d 0.0% 0.0% 0.0% 100% 0.0% 0.0% 0.0% 0.0% let-7e 0.0% 0.0% 0.0% 0.0% 100% 0.0% 0.0% 0.0% let-7f 1.0% 0.0% 0.0% 0.0% 0.0% 100% 0.0% 0.0% let-7g 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 100% 4.0% let-71 0.0% 1.0% 0.0% 0.0% 0.0% 0.0% 0.0% 100% miRNA real-time RT-PCR assay let-7a let-7b let-7c let-7d let-7e let-7f let-7g let-71 let-7a 100% 1.1% 1.1% 0.5% 3.1% 0.1% 0.0% 0.0% let-7b 0.0% 100% 1.8% 0.0% 0.0% 0.0% 0.0% 0.0% let-7c 0.1% 0.5% 100% 0.0% 2.4% 0.0% 0.0% 0.0% let-7d 0.4% 0.1% 0.00% 100% 0.0% 0.0% 0.0% 0.0% let-7e 0.2% 0.1% 0.0% 0.0% 100% 0.0% 0.0% 0.0% let-7f 1.0% 0.6% 0.1% 0.1% 0.0% 100% 0.1% 0.1% let-7g 0.0% 0.6% 0.2% 0.1% 0.0% 0.0% 100% 0.2% let-71 0.0% 0.1% 0.0% 0.0% 0.0% 0.0% 0.1% 100% miRNA real-time RT-PCR assay let-7a let-7b let-7c let-7d let-7e let-7f let-7g let-71 let-7a 100% 2.0% 17.0% 4.0% 4.0% 2.0% 1.0% 2.0% let-7b 1.0% 100% 4.0% 1.0% 1.0% 1.0% 1.0% 1.0% let-7c 0.0% 8.0% 100% 0.0% 0.0% 0.0% 0.0% 0.0% let-7d 2.0% 2.0% 5.0% 100% 1.0% 0.0% 0.0% 0.0% let-7e 1.0% 0.0% 0.0% 0.0% 100% 0.0% 0.0% 0.0% let-7f 6.0% 3.0% 5.0% 3.0% 3.0% 100% 2.0% 3.0% let-7g 0.0% 0.0% 1.0% 0.0% 0.0% 1.0% 100% 4.0% let-71 0.0% 3.0% 0.0% 0.0% 0.0% 0.0% 2.0% 100%

EPIK Supplier Q Supplier T Supplier E

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Reproducibility

Profiling 200 miRNAs from 30 cancer sera over a year in two independent laboratories (R2> 0.95)

Comparison of 200 miRNAs in 30 assays

Measurements taken December 2012 Measurements taken September 2013

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Comparison

RT Input 12 ml serum RNA (=84 ml of serum) RNA input per qPCR reaction 0.41 ml serum RNA

EPIK Biofluid miRNA Panel Supplier E Serum/Plasma Panel

The EPIK Biofluids miRNA Panel Assay (176 miRNAs) was compared to a Serum/Plasma Panel from Supplier E (179 miRNAs) that has a very similar miRNA candidates. The results show earlier Ct values with the EPIK assay, indicating greater sensitivity.

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Advantages

Feature Benefit Advantage

No universal primers Increases specificity Efficiently discrimination of highly homologous miRNA family members Ultra-fast detection Reduces the time to results Allows higher throughput and frees up time to do other work Starting from 1pg of total RNA Can use less or more dilute starting material Important when the source is limited, such as with biopsy tissue

• r dilute, such as in whole blood

samples Detect high and low expression miRNAs over 7 logs of dynamic range All miRNAs can be found from the one sample Saves time, reduces sample volumes and variability All-in-one Designed to complement the RT and SensiSMART™ SYBR mix Can be used directly without further

• ptimization needed, reducing

setup time and saving cost

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Kits

EPIK Cancer miRNA Panel Assay: 352 of the most commonly found miRNAs that could provide insights into cancer pathogenesis, drug response and recurrence. 4 plates x 2 assays per kit. EPIK Stem Cell miRNA Panel Assay: 176 of the most commonly found miRNAs that are associated with induction, maintenance, differentiation and reprogramming of stem cells. 2 plates x 2 assays per kit. EPIK Biofluid miRNA Panel Assay: 176 of the most commonly found miRNAs are regarded as a promising new class of biomarkers and a novel means of extra-cellular communications. 2 plates x 2 assays per kit. EPIK Select Assay: 389 miRNAs (from the miRNAs in the panels above) and an additional ~441 individual wet lab validated miRNAs (over 830 miRNAs in total) are available for further studies. 100 x 4 miRNAs per kit.

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Ordering information

EPIK™ miRNA Panel Assays Size

• Cat. #

EPIK Cancer miRNA Panel Lo-ROX Plate 0.1Y 8 Plates BIO-66031 EPIK Cancer miRNA Panel Hi-ROX Plate 0.1Y 8 Plates BIO-66032 EPIK Cancer miRNA Panel Lo-ROX Plate 0.2Y 8 Plates BIO-66033 EPIK Cancer miRNA Panel Hi-ROX Plate 0.1X 8 Plates BIO-66040 EPIK Stem Cell miRNA Panel Lo-ROX Plate 0.1Y 4 Plates BIO-66034 EPIK Stem Cell miRNA Panel Hi-ROX Plate 0.1Y 4 Plates BIO-66035 EPIK Stem Cell miRNA Panel Lo-ROX Plate 0.2Y 4 Plates BIO-66036 EPIK Stem Cell miRNA Panel Hi-ROX Plate 0.1X 4 Plates BIO-66041 EPIK Biofluid miRNA Panel Lo-ROX Plate 0.1Y 4 Plates BIO-66037 EPIK Biofluid miRNA Panel Hi-ROX Plate 0.1Y 4 Plates BIO-66038 EPIK Biofluid miRNA Panel Lo-ROX Plate 0.2Y 4 Plates BIO-66039 EPIK Biofluid miRNA Panel Hi-ROX Plate 0.1X 4 Plates BIO-66042

EPIK™ miRNA Select Assays Size

• Cat. #

EPIK miRNA Select Hi-ROX Kit 4 x 100 reactions BIO-66046 EPIK miRNA Select Lo-ROX Kit 4 x 100 reactions BIO-66045

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Additional information

• workflow for individual miRNAs and plates

Step 1: cDNA synthesis Step 2: qPCR Step 3: Data analysis

• Excel workbook or online portal

30 min 42 oC 5 min 90 oC 10 min 90 oC 5 min 40 oC 10 s 95 oC 30 s 60 oC

EPIK 2 hours

x40 60 min 42 oC 5 min 90 oC 10 min 90 oC 10 s 95 oC 60 s 60 oC x40

Supplier E 3 hours

35 min 75 min 65 min 120 min