权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Highly accurate small-RNA sequencing of single cells (RealSeq-SC)

单细胞高精度小 RNA 测序 (RealSeq-SC)

基本信息

批准号：
9406996
负责人：
Sergio Barberan-Soler
金额：
$ 28.12万
依托单位：
SOMAGENICS, INC.
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-07-10 至 2019-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9406996
关键词：
Adverse effects Affect Applications Grants Automation Biotechnology Breast Cancer cell line Buffers Cells Chemicals Complementary DNA Coupled Cytolysis Detection Development Digestion Dimerization Evolution Excision Gene Expression Gene Expression Regulation Generations Genetic Transcription Goals Health Heterogeneity High-Throughput Nucleotide Sequencing Homeostasis Individual Libraries Ligation Liquid substance Malignant Neoplasms Measures Mediating Messenger RNA Methods MicroRNAs Modernization Modification Molecular Monitor Noise Oligonucleotides Phase Play Population Preparation Protocols documentation RNA RNA library Reaction Reporting Research Reverse Transcription Ribonuclease H Ribosomal RNA Role Sampling Scheme Small Business Innovation Research Grant Small RNA Splint Device System Technology Testing Transcript Tube base circular RNA design dimer innovation neoplastic cell next generation sequencing novel strategies pre-miRNA repaired single cell analysis single cell sequencing single cell technology transcriptome transcriptomics tumor progression

项目摘要

Abstract 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.

摘要