Recovery and sequence validation of the histological signal following in situ RT-PCR localization of plant gene transcripts

Abstract

An essential facet of functional genomics is an efficient means of examining gene expression at single-cell resolution. We previously developed a high-throughput RT-PCR-based method for in situ localization of gene transcription products. As in all situ methods, it is assumed that the observed signal truly corresponds to the intended target, but this can be prone to false positives. Here we show that the labeled PCR product responsible for the actual histological signal may be recovered and directly analyzed using our method, thereby validating the histological result. We demonstrate that in situ reaction products for abundant (18S rRNA) and rare (Mt-ccs52, which encodes a cell cycle regulator) transcripts can be resolved by means of electrophoresis, blotting, and direct DNA sequencing. Using primers that span anMt-ccs52 intron and DNase treatment, we confirmed that the signal being detected was ultimately derived from mRNA. The ability to experimentally establish the specificity of an in situ signal after the fact enhances the high-throughput capability of the in-well RT-PCR method and further increases the utility of this technique as a powerful tool for functional genomics studies.