Chimeric probe-mediated ribonuclease protection assay for molecular diagnosis of mRNA deficiencies

Investigations throughout the last decade have established that cytoskeleton integrity ensures red cell deformability and mechanical stability, and that defects in one of the skeletal components usually result in more or less severe hemolytic anemias. Although a large number of molecular defects have been identified to date, many others still bypass fast and commonly used methods, such as SSCP and DGGE, mostly because of a subtle change in mRNA transcription level or a complex interaction leading to the loss of other components. We describe a ribonuclease protection assay based on a simultaneous quantification of two cytoskeletal transcripts, using a chimeric probe, emphasizing the value of a nonspecific bridging sequence, inserted between the two specific probe sequences. It is anticipated that this powerful and reliable procedure would be an additional tool in the methodology array used for screening cytoskeletal inherited abnormalities.