High throughput cellular localization of specific plant mRNAs by liquid-phase in situ reverse transcription-polymerase chain reaction of tissue sections.
Advances in high throughput DNA sequencing and bioinformatic gene discovery far outpace our ability to analyze gene function, necessitating development of more efficient means to examine expression at the cellular level. Here we present a polymerase chain reaction-based method to detect mRNA species in situ in which essentially all of the steps are carried out in liquid phase in a 96-well microtiter tray and only the final signal detection is performed on a microscope slide. We demonstrate the sensitivity of the method by the cellular localization of mRNA for the Tkn2 transcription factor in a wide variety of plant tissues, and its selectivity in discriminating a single gene family member by the in situ localization of rbcs3 transcripts. Furthermore, we demonstrate the utility of the in-well in situ method in detecting FDL and IFL1 transcripts in Arabidopsis sections, thus establishing the method as a tool to determine spatial expression pattern of sequences obtained from genomic sequencing projects. Being amenable to robotic processing, in-well in situ reverse transcription-polymerase chain reaction permits a great enhancement in the number of tissue samples that can be processed. Consequently, this method may become a powerful tool for functional genomics studies, permitting the cellular site of transcription of large numbers of sequences obtained from databases to be rapidly established.
Department of Plant Pathology, North Carolina State University, Raleigh, North Carolina 27695, USA.
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D017360:Arabidopsis / Q000033:anatomy & histology / Q000235:genetics*; D020869:Gene Expression Profiling / Q000379:methods; D017343:Genes, Plant; D018551:Solanum lycopersicum / Q000033:anatomy & histology / Q000235:genetics*; D008857:Microscopy, Interference; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
D012333:RNA, Messenger