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Two methods for full-length RNA sequencing for low quantities of cells and single cells


Departments of Genetics , Yale University School of Medicine, New Haven, CT 06520;Weill Cornell Medical College, New York, NY 10065;Departments of Genetics , Yale University School of Medicine, New Haven, CT 06520,Department of Cell Biology, Second Military Medical University, Shanghai 200433, China;Departments of Genetics , Yale University School of Medicine, New Haven, CT 06520;Departments of Genetics , Yale University School of Medicine, New Haven, CT 06520,Jiangsu University Affiliated Hospital, Zhenjiang, Jiangsu 212001, China;Departments of Genetics , Yale University School of Medicine, New Haven, CT 06520;Departments of Genetics , Yale University School of Medicine, New Haven, CT 06520;Department of Genetics, Stanford University, Palo Alto, CA 94305;Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100005, China,Departments of Anesthesiology, Yale University School of Medicine, New Haven, CT 06520;Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100005, China;Departments of Genetics , Yale University School of Medicine, New Haven, CT 06520;Department of Genetics, Stanford University, Palo Alto, CA 94305;Weill Cornell Medical College, New York, NY 10065;Departments of Genetics , Yale University School of Medicine, New Haven, CT 06520;%The ability to determine the gene expression pattern in low quantities of cells or single cells is important for resolving a variety of problems in many biological disciplines. A robust description of the expression signature of a single cell requires determination of the full-length sequence of the expressed mRNAs in the cell, yet existing methods have either 3' biased or variable transcript representation. Here, we report our protocols for the amplification and high-throughput sequencing of very small amounts of RNA for sequencing using procedures of either semirandom primed PCR or phi29 DNA polymerase-based DNA amplification, for the cDNA generated with oligo-dT and/or random oligonucleotide primers. Unlike existing methods, these protocols produce relatively uniformly distributed sequences covering the full length of almost all transcripts independent of their sizes, from 1,000 to 10 cells, and even with single cells. Both protocols produced satisfactory detection/coverage of the abundant mRNAs from a single K562 erythroleukemic cell or a single dorsal root ganglion neuron. The phi29-based method produces long products with less noise, uses an isothermal reaction, and is simple to practice. The semirandom primed PCR procedure is more sensitive and reproducible at low transcript levels or with low quantities of cells. These methods provide tools for mRNA sequencing or RNA sequencing when only low quantities of cells, a single cell, or even degraded RNA are available for profiling.......

【作者名称】: Xinghua Pan, Russell E. Durrett, Haiying Zhu, Yoshiaki Tanaka, Yumei Li, Xiaoyuan Zi
【作者单位】: Departments of Genetics , Yale University School of Medicine, New Haven, CT 06520, Weill Cornell Medical College, New York, NY 10065, Departments of Genetics , Yale University School of Medicine, New Haven, CT 06520,Department of Cell Biology, Second Military Medical University, Shanghai 200433, China, Departments of Genetics , Yale University School of Medicine, New Haven, CT 06520, Departments of Genetics , Yale University School of Medicine, New Haven, CT 06520,Jiangsu University Affiliated Hospital, Zhenjiang, Jiangsu 212001, China, Departments of Genetics , Yale University School of Medicine, New Haven, CT 06520
【关 键 词】: RNA-seq, transcriptome amplification, full-length mRNA, single-cell analysis
【期刊名称】: Proceedings of the National Academy of Sciences of the United States of America
【期刊论文数据库】: [DBS_Articles_01]
【期刊论文编号】: 107,248,791
【摘要长度】: 2,894
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