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 developmen t 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 ph ase in a 96-well microtiter tray and only the final signal detection is per formed 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 sin gle gene family member by the in situ localization of rbcs3 transcripts. Fu rthermore, we demonstrate the utility of the in-well in situ method in dete cting FDL and IFL1 transcripts in Arabidopsis sections, thus establishing t he method as a tool to determine spatial expression pattern of sequences ob tained from genomic sequencing projects. Being amenable to robotic processi ng, 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 genomi cs studies, permitting the cellular site of transcription of large numbers of sequences obtained from databases to be rapidly established.