Generation of amplified RNAs and cDNA libraries from single mammalian cells.
With the near completion of the human genome sequencing effort, it is now possible to analyze the expression of the entire human gene complement. However, a major obstacle in performing such analysis is the ability to successfully generate enough cDNA or amplified RNA from a limited number of cells, such as biopsies, blood smears, cells obtained by laser capture microscopy, and preimplantation embryonic cells and germ cells. Because these samples yield extremely small amounts of RNA, reproducible methods are needed to amplify this RNA while maintaining the original message profile. A detailed description is given for generating pools of cDNA libraries containing a high proportion of cDNAs enriched with 5'-coding sequences from as little as 1 ng of total RNA using a modified switching mechanism at 5' end of RNA transcript protocol. In addition, the T7-promoter-linked double-stranded cDNAs can be in vitro transcribed linearly using T7-RNA polymerase to generate amplified RNA that is mRNA derived. The cDNA pools can be used directly for gene-specific reverse transcriptase polymerase chain reaction or processed for ligation into vectors of choice whereas the amplified RNA can be used for microarray-based expression profiling.
Adjaye
James
J
Department of Vertebrate Genomics, Max Planck Institute for Molecular Genetics, Berlin, Germany.
D002477:Cells / Q000737:chemistry / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods*; D015723:Gene Library; D006801:Humans; D012313:RNA / Q000096:biosynthesis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014764:Viral Proteins / Q000235:genetics
D014764:Viral Proteins; D012313:RNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases