A large-scale whole-mount in situ hybridization system: Rapid one-tube preparation of DIG-labeled RNA probes and high throughput hybridization using 96-well silent screen plates

Abstract

Recent progress in multiple and automated-sequencing technology allows large-scale random cDNA sequencing, the so-called EST project, in various fields. In addition to the EST collection, the cDNA project requires analysis of spatiotemporal patterns of gene expression of a large number of clones by whole-mount in situ hybridization (WISH). To facilitate the multiple WISH procedures, we developed a protocol for rapid and uniform synthesis of multiple probes and multi-well based WISH processing. A DIG-labeled RNA probe for WISH was synthesized from a PCR-amplified template which contained an RNA promoter. All reactions of PCR and subsequent RNA synthesis were performed in a single tube by sequential addition of the reagents without phenol extraction or ethanol precipitation steps. An RNA probe was purified and condensed by a centrifugal ultrafilter to achieve high and stable purification efficiency. WISH of 96 samples were performed simultaneously in a 96-well plate attached to silent screen filters that were connected with a vacuum exhausting system. These processes eliminated the labor-intensive steps of WISH and provided opportunities to search for novel genes.