Analysis of gene expression in transgenic plants.
Recent years have seen a huge increase and improvement in techniques for analysis of transgene expression in plants. The analysis of RNA frequently provides a vital link between changes in enzyme levels and/or metabolites and the phenotype. This chapter focuses on RNA-based techniques for the analysis of transgene expression, beginning with the extraction of RNA and its evaluation in terms of purity and integrity by spectrophotometry and gel electrophoresis, respectively. Common methods of transcript analysis by Northern and dot-blot hybridizations using nonradioactive probing methods are described. A protocol for reverse-transcriptase polymerase chain reaction (RT-PCR) as a method of establishing transgene expression qualitatively, as well as a procedure for quantitative RT-PCR for comparing relative abundance of transcript levels of two or more genes are described. Lastly, a protocol for localization of RNA transcripts within tissues by in situ hybridization is included.
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Andrew F
AF
Department of Plant Biology, University of New Hampshire, Durham, USA.
Minocha
Subhash C
SC
D015152:Blotting, Northern / Q000379:methods; D005786:Gene Expression Regulation / Q000235:genetics*; D017403:In Situ Hybridization; D030821:Plants, Genetically Modified / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014158:Transcription, Genetic
D012333:RNA, Messenger; D011994:Recombinant Proteins