SBIR-STTR Award

The goal of this grant application is to develop the first commercially available library preparation kit for profiling small RNAs from single cells using NGS methods. Single cell analyses of mRNA have allowed the identification of crucial differences between cells that were otherwise considered identical. These findings have shown that there is intrinsic “noise” in the regulation of gene expression that plays an important role in determining cell fates. Unfortunately, there is currently a lack of information about the cell-to-cell variability of levels of ?small RNAs, including microRNAs. Indeed, there is no commercially available library preparation kit for small RNAs that can profile single cells. We propose to further develop our library preparation technology, RealSeq®-AC, to be able to quantify small RNAs from single cells. RealSeq®-AC uses a scheme involving a single combo-adapter and circularization that accurately quantifies over 75% of all miRNAs detected, compared to ~35% from the best competitor kit. To adapt this technology for single-cell sequencing we will test three separate strategies to dramatically reduce the presence of adapter-dimers in the library, as well as possible use of miRNA pre-amplification to reduce the number of PCR cycles needed. We will develop a protocol that performs all steps from cell lysis to final purification of amplified libraries in a single tube. This technology will allow the accurate quantification of small RNAs from single cells.

Public Health Relevance Statement:
Health relatedness narrative Transcriptomic analysis of single cells has shown that there is a high cell-to-cell variability in gene expression among apparently identical cells in a population. However, a similar analysis is not currently possible for levels of small RNAs, which play key roles in regulating gene expression. The goal of this grant application is to develop the first commercially available library preparation kit for profiling microRNAs from single cells using modern high-throughput sequencing methods. Understanding how individual cells vary in their small RNA profiles is of central importance to understanding cancer progression and the evolution of malignant tumor cells.

Project Terms:
Adverse effects; Affect; Applications Grants; Automation; base; Biotechnology; Breast Cancer cell line; Buffers; Cells; Chemicals; circular RNA; Complementary DNA; Coupled; Cytolysis; design; Detection; Development; Digestion; dimer; Dimerization; Evolution; Excision; Gene Expression; Gene Expression Regulation; Generations; Genetic Transcription; Goals; Health; Heterogeneity; High-Throughput Nucleotide Sequencing; Homeostasis; Individual; innovation; Libraries; Ligation; Liquid substance; Malignant Neoplasms; Measures; Mediating; Messenger RNA; Methods; MicroRNAs; Modernization; Modification; Molecular; Monitor; neoplastic cell; next generation sequencing; Noise; novel strategies; Oligonucleotides; Phase; Play; Population; pre-miRNA; Preparation; Protocols documentation; Reaction; repaired; Reporting; Research; Reverse Transcription; Ribonuclease H; Ribosomal RNA; RNA; RNA library; Role; Sampling; Scheme; single cell analysis; single cell sequencing; single cell technology; Small Business Innovation Research Grant; Small RNA; Splint Device; System; Technology; Testing; Transcript; transcriptome; transcriptomics; Tube; tumor progression

The goal of this grant application is to develop the first commercially available library preparation kit for profiling small RNAs from single cells using NGS methods. Single-cell analyses of mRNA have allowed the identification of crucial differences between cells that were otherwise considered identical. These findings have shown that there is intrinsic “noise” in the regulation of gene expression within a population of cells that plays an important role in determining cell fates. Unfortunately, there is currently a lack of information about the cell-to-cell variability of levels of microRNAs that as gene expression regulators may also play a critical role. Indeed, there is no commercially available library preparation kit for miRNAs and other small RNAs that can profile single cells. We propose to quantify miRNAs from single cells using an advanced, proprietary “low input single adapter and circularization” technology that allows sensitive and unbiased detection. The core single adapter and circularization technology, for higher input quantities, demonstrated unbiased detection of over 70% of all miRNAs in a benchmark Universal miRNA pool, compared to ~35% from the best competitor kit. We have further developed this technology for single-cell analysis by creating a novel “low input version” that retains the detection accuracy even at single- cell levels. Data from our Phase I studies show that this “low input adapter” minimizes dropout events (a critical and common problem in single cell analysis) by increasing the efficiency of miRNA detection. Another major obstacle for single-cell miRNA sequencing is formation of adapter-dimers lacking miRNA inserts during library preparation that critically reduces the amount of useful miRNA sequencing reads. We employ three separate strategies to dramatically reduce the presence of adapter-dimers in the library. Also, our protocol performs all steps from cell lysis to final purification of amplified libraries in a single tube to reduce loss of miRNA from single-cells and to reduce the possibility of contamination of single-cell samples by environmental RNA. In Phase I we demonstrated proof-of-principle by detecting small RNAs from single-cells for three different cell lines. In Phase II, we will further develop and optimize our technology to significantly increase sensitivity and detection accuracy of miRNAs and other small RNAs from single cells for commercial viability. We will also develop a kit for single-cell small RNA-seq library preparation (RealSeq-SC).

Public Health Relevance Statement:
Health relatedness narrative Transcriptomic analysis of single cells has shown that there is a high cell-to-cell variability in gene expression among apparently identical cells in a population. However, a similar analysis is not currently possible for levels of small RNAs, which play key roles in regulating gene expression. The goal of this grant application is to develop the first commercially available library preparation kit for profiling microRNAs from single cells that enables detection of very early changes in development of individual tumor cells and eventually could be used for early-stage cancer diagnostics and for more effective, personalized therapeutic development.

Project Terms:
Applications Grants; base; Benchmarking; Blood specimen; Breast; Breast Cancer cell line; Cancer Diagnostics; Cell Line; Cells; Chemicals; circulating biomarkers; cost effective; Cytolysis; Data; design; Detection; Development; dimer; Dropout; Event; experimental study; Gene Expression; Gene Expression Regulation; Genome; Goals; Grant; Health; Individual; Lead; Libraries; Ligation; magnetic beads; Manufacturer Name; Messenger RNA; Methods; Microfluidics; MicroRNAs; miRNA expression profiling; Modification; Neoplasm Circulating Cells; neoplastic cell; next generation sequencing; Noise; novel; novel strategies; Oligonucleotides; personalized therapeutic; Phase; phase 1 study; Play; Population; Preparation; Protocols documentation; Publishing; Reaction; Reagent; Research Personnel; RNA; Role; Sampling; Signal Transduction; single cell analysis; single cell technology; single-cell RNA sequencing; Small RNA; Sorting - Cell Movement; Technology; therapeutic development; transcriptome; transcriptome sequencing; transcriptomics; Tube; Whole Blood