DETECTION OF BETA-ACTIN MESSENGER-RNA BY RT-PCR IN NORMAL AND REGENERATING CHICKEN COCHLEAE

Reverse transcriptase polymerase chain reaction (RT-PCR) was used to show that beta-actin RNA levels can be detected in total RNA isolations from as few as two cochleae. In functionally mature chicken cochleae, a low homeostatic level of beta-actin message should be expressed in order to synthesize enough actin to maintain the stereocilia, cuticular plate, junctional complexes of hair cells and the cytoskeletal components of the supporting cells. The RT-PCR product obtained has been characterized by size, restriction digest analysis, and DNA sequencing analysis. These procedures have confirmed that the product is amplified from a chicken beta-actin mRNA target. Subsequently, semi-quantitative RT-PCR techniques were used to demonstrate an upregulation of beta-actin mRNA transcription levels in the cells of the basilar papilla during regeneration following damage from acoustic overstimulation. These studies suggest that RT-PCR can be utilized for analysis of limited quantities of tissue such as that found in the chicken cochlea and indicate promise for further qualitative and quantitative studies on the molecular mechanisms of hair cell transduction and regeneration.