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SANTA CRUZ, CA, United States

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 225.00K | Year: 2014

Our goal in this proposal is to develop a novel method for the direct detection of miRNAs in serum and plasma without requiring isolation of total RNA. Distinct expression profiles of microRNAs (miRNAs) have recently been associated with cancer and other diseases, implying that miRNAs could serve as diagnostic biomarkers for these diseases. Effective biomarkers could facilitate early detection of cancer, leading to improved therapeutic outcomes, and aid in monitoring progression and response to therapy. TheRT-qPCR assays currently employed to quantify circulating miRNAs are hampered by problems associated with their requirement for isolating total RNA from plasma or serum prior to miRNA quantification. RNA is typically isolated by spin-column purification or phenol extraction and ethanol precipitation. Both methods suffer from inconsistent miRNA recovery and the lack of an internal reference miRNA for data normalization. Moreover, current RNA isolation procedures do not completely remove inhibitors of enzy

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 269.38K | Year: 2014

DESCRIPTION (provided by applicant): This proposal addresses the problem of bias in the expression profiling of microRNAs (miRNAs) and other small RNAs by next-generation sequencing (NGS). Because dysregulation of miRNA expression has been implicated in cancer and certain other diseases, accurate expression profiling of all miRNA sequences is important for understanding miRNA biology and for development of new biomarkers and therapeutic targets. NGS is currently the most comprehensive approach for digital gene expression profiling, and is used for the discovery of novel miRNA sequences, identification of sequence variants, and the quantification of known miRNAs. Unlike other expression profiling platforms such as microarrays or RT-qPCR, NGS combines unlimited multiplexing capability, single-molecule sensitivity, a superior dynamic range, and true sequence specificity without requiring prior knowledge of miRNA sequences. However, NGS expression profiling data underestimate the amount of many miRNAs in a s

Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 287.79K | Year: 2016

DESCRIPTION provided by applicant Quantification of gene expression in formalin fixed paraffin embedded FFPE tissue samples is important for the discovery and validation of cancer biomarkers for tumor classification and to assess progress during cancer treatment Because RT qPCR assays are very sensitive and sequence specific they are currently preferred for mRNA expression profiling in FFPE tissues as well as for validation of data obtained by other expression profiling methods such as microarrays and sequencing RNA seq However use of these methods to analyze FFPE samples is constrained by the RNA fragmentation that occurs in these samples and limits the sensitivity and reproducibility of these assays To overcome this problem we proposed a novel method for assaying mRNA fragments in FFPE samples called mR FQ mRNA Fragment Quantification In Preliminary Studies we developed a mR FQ prototype which can work with very short mRNA fragments of nt and demonstrated its superior sensitivity over the TaqMan RT qPCR method in quantifying two model mRNAs from FFPE samples We analyzed HER a breast cancer biomarker and GAPDH internal reference mRNAs in total RNA isolated from breast cancer and prostate cancer FFPE samples In Phase I we plan to i validate the mR FQ method using a larger number of target mRNAs and more FFPE samples ii further optimize mR FQ iii demonstrate that mR FQ reliably quantifies mRNAs in FFPE samples containing highly fragmented mRNAs that are not detectable by currently leading RT qPCR methods run in parallel iv determine the maximum mRNA fragmentation level detectable by mR FQ In Phase II we will move towards commercialization by designing mR FQ assays for BC biomarker candidates and validating them on FFPE samples having a wide range of RNA fragmentation levels with focus on currently unusable samples those containing highly fragmented RNA that cannot be assayed by standard RT qPCR methods mR FQ and RNA seq analyses will be compared and we expect that mR FQ will be able to validate RNA seq results since both methods can work with highly fragmented RNA This will allow researchers to include a wider range of FFPE samples into retrospective studies for the development and validation of improved breast as well as other types of cancer diagnostics and treatment prognostics PUBLIC HEALTH RELEVANCE This project develops new technology for studying gene expression archived tissue specimens from cancer patients We plan to use it to develop better biomarkers for breast cancer the most common cancer among women About women were diagnosed and over died from the disease last year in the US alone The goal of this grant application is to develop a method for quantitation of fragmented mRNAs from standard histology specimens FFPE blocks that will provide superior sensitivity and accuracy at a reasonable cost to facilitate research in and diagnosis of breast cancer as well as treatment prognosis

Methods, compositions, and kits comprising target-specific oligonucleotides (TSOs) are disclosed herein. Methods, compositions, and kits comprising target-specific oligonucleotides (TSOs) can be used to attach adapters and/or linkers to target RNAs. Methods, compositions, and kits comprising target-specific oligonucleotides (TSOs) can be used in reactions, including, but not limited to, ligation reactions, amplification reactions, and sequencing reactions. Additionally, methods, compositions, and kits comprising target-specific oligonucleotides (TSOs) can be used for reducing and/or preventing the formation of secondary structures in target RNAs. These methods, compositions, and kits can also find use in a number of applications, for example, any application that benefits from stabilizing primary RNA structure, such as detecting and quantifying target RNAs in a sample, in the construction of small RNA libraries, in microarray and RT-qPCR applications, etc.

Somagenics, Inc. | Date: 2015-08-25

Currently, the circularization of small RNAs is broadly regarded as an obstacle in ligation-related assays and explicitly avoided while short lengths of linear RNA targets is broadly recognized as a factor limiting use of conventional primers in PCR-related assays. In contrast, the disclosed invention capitalizes on circularization of small RNA targets or their conjugates with oligonucleotide adapters. The circular RNA templates provide amplification of the target sequences via synthesis of multimer nucleic acids that can be either labeled for direct detection or subjected to PCR amplification and detection. Structure of small circular RNAs and corresponding multimeric nucleic acids provide certain advantages over current methods including flexibility in design of conventional RT and PCR primers as well as use of 5-overlapping dimer-primers for efficient and sequence-specific amplification of short target sequences. Our invention also reduces number of steps and reagents while increasing sensitivity and accuracy of detection of small RNAs with both 2OH and 2-OMe at their 3 ends. Our invention increase sensitivity and specificity of detection of microRNAs and other small RNAs with both 2OH and 2-OMe at their 3 ends while allowing us to distinguish these two forms from each other.

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