Somagenics, Inc.

Realseq Biosciences, Inc.

Sergei A Kazakov

Brian H Johnston

Anne Dallas

Pavan Kumar

Heini Ilves

Qing Ge

Sergio Barberan-Soler

Catharina Casper-Lindley

Disclosed herein are methods and compositions for the detection of small RNAs in a sample. The methods and compositions disclosed herein may be used for preparing sequencing libraries of the small RNAs, fragments of RNAs and DNAs.

Methods of library construction for polynucleotide sequencing

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.

Methods of constructing small RNA libraries and their use for expression profiling of target RNAs

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 2&#x2032;OH and 2&#x2032;-OMe at their 3&#x2032; ends. Our invention increase sensitivity and specificity of detection of microRNAs and other small RNAs with both 2&#x2032;OH and 2&#x2032;-OMe at their 3&#x2032; ends while allowing us to distinguish these two forms from each other.

Methods and compositions for detection of small RNAs

Disclosed herein are compositions and methods for the processing, amplification and detection of polynucleotides using target-specific oligonucleotides (TSOs). Hybridization of TSOs to target polynucleotides guides target processing into and purification of small target fragments that then can be amplified and detected with high sensitivity and reproducibility. The method is specifically beneficial for highly degraded polynucleotides found in biological samples.

Methods and compositions for detecting polynucleotides and fragments thereof

Methods and compositions for detection of small RNAS

Methods, compositions, and kits that include small hairpin RNA (shRNA) useful for inhibition of gene expression, such as viral-mediated gene expression, are described.

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San Jose, CA, United States of America

Sergei A Kazakov