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

EPIK ™ miRNA Panel and Individuals Assays Better by Design www.bioline.com

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 - Insulin secretion - Stem cell differentiation - Cholesterol metabolism - Hematopoiesis - Aging - Immune responses - Hypoxia - Viral replication - Cardiac and skeletal muscle development - Embryogenesis - Neurogenesis www.bioline.com www.bioline.com

Why use qPCR qPCR Microarray Sequencing 1 – 2 weeks Throughput time ~6 hours ~2 days Total RNA required 500 ng 100-1,000 ng 500-5,000 ng Five or more Dynamic range Six orders of Four orders of orders of detected magnitude magnitude magnitude Infrastructure and technical Few Moderate Substantial requirements www.bioline.com www.bioline.com

SYBR Based qPCR miRNA The most commonly used universal tailing method, reduces specificity 1 Some suppliers using modified RNA nucleotides (LNA) to increase hybridization properties, however, they can display poorer amplification efficiencies, reducing sensitivity 2 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). www.bioline.com www.bioline.com

Probe Based qPCR miRNA The use of miRNA-specific forward and reverse primers offers 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 www.bioline.com www.bioline.com

New Probe Based qPCR miRNA Mature microRNA Poly A Tailing The addition of adaptors on both ends has an advantage in terms of the specificity as the probe has more space, Ligation additionally an amplification step increases sensitivity Universal RT However the TaqMan probes still need to be designed for each miRNA and so is much miR-Amp more expensive. The extra steps also makes the whole process more time qPCR consuming and more prone to mistakes www.bioline.com www.bioline.com

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 www.bioline.com www.bioline.com

Feature comparison Supplier T Bioline 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 100ng 500ng 10ng 1mg 100ng reaction 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 6 m l for RNA in 5 m l for RNA in 2 m l for RNA in 15 m l for RNA in 2 m l for RNA in Volume of RNA/total a 20 m l reaction a 15 m l reaction a 5 m l reaction a 20 m l reaction a 10 m l reaction volume RT reaction Components supplied In 1 kit As 3 Kits As 3 Kits As 3 Kits As 3 Kits Company confidential www.bioline.com www.bioline.com

Sensitivity Ct value (10 8 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 Comparison of five miRNAs with increasing AT concentrations 40 Assay Efficiency: 99.0% 30 10 8 Ct 20 100 copies 10 y = -3,332x + 39,348 copies R² = 0,9997 0 NTC 0 5 10 Log (copies of template) The results illustrate the sensitivity (10 8 to 100 copies) and efficiency (99.97%) of the assay, allowing the detection of miRNAs at varying expression levels, including low expressers. www.bioline.com www.bioline.com

Sensitivity EPIK Supplier E Ensuring that 10 8 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. www.bioline.com www.bioline.com

Specificity Relative detection across the Let-7 miRNA family shows at least 50-fold specificity and virtually no cross-reaction. 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% 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 A C A G U U 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. www.bioline.com www.bioline.com

Specificity comparison EPIK Supplier Q miRNA real-time RT-PCR assay miRNA real-time RT-PCR assay EPIK let-7a let-7b let-7c let-7d let-7e let-7f let-7g let-71 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-7a 100% 1.1% 1.1% 0.5% 3.1% 0.1% 0.0% 0.0% let-7b 0.0% 100% 2.2% 0.0% 0.0% 0.0% 0.0% 0.0% let-7b 0.0% 100% 1.8% 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-7c 0.1% 0.5% 100% 0.0% 2.4% 0.0% 0.0% 0.0% let-7d 0.4% 0.0% 0.1% 100% 0.0% 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 0.1% 0.0% 0.0% 0.0% 100% 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 0.1% 0.0% 0.0% 0.0% 0.0% 100% 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% 0.0% 0.0% 0.0% 0.0% 0.0% 100% 0.0% let-7g 0.0% 0.6% 0.2% 0.1% 0.0% 0.0% 100% 0.2% let-71 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 100% let-71 0.0% 0.1% 0.0% 0.0% 0.0% 0.0% 0.1% 100% Supplier T Supplier E miRNA real-time RT-PCR assay miRNA real-time RT-PCR assay let-7a let-7b let-7c let-7d let-7e let-7f let-7g let-71 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-7a 100% 2.0% 17.0% 4.0% 4.0% 2.0% 1.0% 2.0% let-7b 0.0% 100% 3.0% 0.0% 0.0% 0.0% 0.0% 0.0% let-7b 1.0% 100% 4.0% 1.0% 1.0% 1.0% 1.0% 1.0% let-7c 1.0% 2.0% 100% 0.0% 0.0% 0.0% 0.0% 0.0% let-7c 0.0% 8.0% 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-7d 2.0% 2.0% 5.0% 100% 1.0% 0.0% 0.0% 0.0% let-7e 0.0% 0.0% 0.0% 0.0% 100% 0.0% 0.0% 0.0% let-7e 1.0% 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-7f 6.0% 3.0% 5.0% 3.0% 3.0% 100% 2.0% 3.0% let-7g 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 100% 4.0% let-7g 0.0% 0.0% 1.0% 0.0% 0.0% 1.0% 100% 4.0% let-71 0.0% 1.0% 0.0% 0.0% 0.0% 0.0% 0.0% 100% let-71 0.0% 3.0% 0.0% 0.0% 0.0% 0.0% 2.0% 100% Company confidential www.bioline.com www.bioline.com

Reproducibility Profiling 200 miRNAs from 30 cancer sera over a year in two independent laboratories (R2> 0.95) Measurements taken December 2012 Measurements taken September 2013 Comparison of 200 miRNAs in 30 assays www.bioline.com www.bioline.com

Comparison EPIK Biofluid miRNA Panel Supplier E Serum/Plasma Panel 12 m l serum RNA (=84 m l of serum) RT Input 0.41 m l serum RNA RNA input per qPCR reaction 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. www.bioline.com www.bioline.com