High-Sensitivity Multiplexed Assay and Associated Bioinformatics Tools for Measuring and Analyzing up to 68 microRNAs from Serum, Plasma, and Urine in 96-Well Format Andrea Bryan, Timothy Erps, Arturo Escajeda, Anthony Fusco, Jeﬀrey Horne, Arben Koshi, Davide Marini, Daniel Pregibon, Conor Raﬀerty, Isaac Stoner, Mike Tackett, Jessica Tytell, Andreas Windemuth Fireﬂy BioWorks, One Kendall Square, 1400W, Cambridge, MA 02139 Abstract We used reference RNA from three tissue types in order to benchmark the Firefly™ microRNA HS assay with existing profiling methodologies, showing that assay correlates well (Pearson > 0.8) with each. Prior to Pearson calculations, samples were first mean normalized (signal for a given target in a sample was divided by the average signal of all targets in that sample). Once target was normalized, signals were sample normalized by dividing the target signal in a given sample by the average signal across samples for that particular target. The Firefly™ HS microRNA assay was also compared to results obtained on the NanoString (NS) system and gave a Pearson correlation of 0.8. We isolated RNA from the serum samples (200ul) of three healthy individuals (A -C) using a modified Trizol-LS protocol (a negative control isolation from water was also performed). The Firefly™ miRNA HS assay was performed to simultaneously quantify 30 miRNAs in each sample with final assay readout on a Guava easyCyte 8HT. The results from duplicate isolations were averaged and mean normalized. The miRNA profiles show a broad range of miRNA levels with robust detection of most targets assayed. Arbitrary Units 100 A B 10 C 1 TLDA Sequencing Micro Array Fireﬂy HS Assay Overview TLDA 1 (0) 0.85 (0.02) 0.83 (0.1) 0.79 (0.14) Sequencing Micro Array 1 (0) 0.81 (0.1) 0.81 (0.08) 1 (0) 0.84 (0.1) Integrated Bioinformatics Fireﬂy HS 1 (0) Reproducibility We performed the assay on total RNA purified from 200ul of human serum by a modified Trizol protocol in quadruplicate in order to assess reproducibility. Targets present at abundances at the middle or top end of the dynamic range (> 10 zeptomoles) give coefficients of variation typically less than 10% (Figure A). Variation increases as signals approach the Limit of Detection but typically remain < 50% (Figure B). A High Sensitivity Technical Replicates (High Abundance Targets) Target and Sample Normalized Signal (AU) Hydrogel particles made using Firefly BioWorks' patented Optical Liquid Stamping are hydrophilic, bio-inert, and flexible. The particles (shown in A) are porous, allowing target molecules to diffuse and bind in a unique nanoscale three-dimensional scaffold. This both maximizes signal and widens the assay's dynamic range. The particles are “barcoded” with different levels of fluorescence in each of tow barcode regions (shown in B), forming up to 70 unique codes. Each uniquely-barcoded particle is then functionalized with an individual probe complementary to the microRNA of interest enabling multiplexing of up to 68 custom targets along with a positive and negative control in each well of a 96-well plate, all with a short 4-hour workflow (shown in C). The Firefly™ HS microRNA Assay uses a unique post-hybridization labeling scheme followed by bias-free PCR with universal primers to selectively amplify only captured targets. Results are read out by flow cytometry and analyzed using our free bioinformatics-enabled software tools. microRNA Proﬁling in Serum 1000 5 4 3 2 1 0 B Target and Sample Normalized Signal (AU) Firefly BioWorks has created a miRNA profiling assay that enables the profiling of up to 96 samples in one afternoon with PCR-level sensitivity. The assay is completely customizable, allowing the detection of up to 68 user-selected miRNAs per sample. The platform is built on barcoded microgels that can be scanned on existing flow cytometers. We demonstrate how this assay can be used for the highly sensitive detection of miRNAs in tissue, plasma and other sample types, generating robust miRNA profiles with as little as 1ng input. We also introduce bioinformatics tools that are integrated with the assay analysis software to cluster co-regulated microRNAs and help identify potentially relevant genes and pathways as well as recommend additional microRNAs for study. Cross-Platform Comparison High Sensitivity Technical Replicates (Lower Abundance Targets) Biomarker discovery and validation typically involves additional challenges in the area of target selection and data analysis. Firefly has created two bioinformatics tools to aid both selection and analysis of microRNA targets: the Firefly™ Discovery Engine and the Firefly™ Analysis Workbench. The web-based Firefly™ Discovery Engine automatically generates a list of the most relevant microRNAs and associated gene targets for any keyword or topic, returning a list of the most published microRNAs for that topic with an annotated list of associated publications. The Firefly™ Analysis Workbench analyzes particle scan data providing means for rapid data analysis and visualization. The Workbench enables streamlined generation of publication-ready heatmaps and bargraphs. Clusters of miRNAs identified in a study are cross-referenced with genomic databases to provide further insghts into associated genes and pathways. Together, this novel combination of bioinformatics tools and multiplexed high-sensitivity assays enables rapid validation and screening of microRNA biomarker signatures from serum, plasma, and urine in high throughput format. 0.14 0.12 0.1 0.08 0.06 0.04 0.02 0 Fireﬂy™ Discovery Engine www.fireflybio.com/search Robustness and Input Amount We demonstrate robust miRNA proﬁling over a broad range of input RNA from 100 down to 1ng. Without normalization (A), signals decrease linearly with decreasing sample input. After Quantile Normalization is applied (B), the proﬁles for all sample input leves are virtually identical. As expected, the correlation between the samples are very strong, with Pearson coeﬃcients >0.95. These results demonstrate the sensitivity and dynamic range of the assay, but also show that the assay is not biased by input amount. Sensitivity Analysis Fireﬂy™ Analysis Workbench A. Non-normalized C. Correlation between input amounts We assessed the sensitivity of the assay using synthetic microRNA spike-ins across a range from 1 attomole (600,000 molecules) to 1 zeptomole (600 molecules). Particles containing a negative control probe, cel-miR-39-3p, were also included in all experimental wells. All target levels assesed yielded detectable signal with excellent linearity across the range tested. Error bars are shown between technical replicates. 100 ng 10 ng 1 ng 100 ng 1 0.9894 0.9574 10 ng 1 ng 1 0.9646 1 100 ng vs 10 ng hsa-mir-141-3p 10000 10000 B. Normalized hsa-mir-143-3p 1000 hsa-mir-203 cel-let-7-5p 100 10 100 Arbitrary Units hsa-mir-210 R² = 0.9894 1000 10 1 0.1 1 0.1 10 100 1000 10000 Integrated Cluster Analysis 0.01 1 0 600 6,000 molecules 60,000 600,000 www.ﬁreﬂybio.com