METHODS FOR QUANTITATIVE-ANALYSIS OF TRANSCRIPTION IN LARVAE AND PREPUPAE

This chapter discusses the methodologies used to study gene activity during the early stages of metamorphosis. It describes the methods currently available for staging third instar larvae and prepupae. Gene activities in these animals are then assessed by isolating RNA from intact animals or dissected organs. Extracted organs can be maintained in culture and treated with hormone in vitro to induce specific responses, after which RNA is isolated and analyzed. Two methods are used to extract RNA from organs or intact animals. Both procedures yield samples that contain both DNA and RNA. As DNA represents only ∼10% of the total nucleic acids, however, it is of no consequence for subsequent Northern blot hybridization analysis. The DNA does not appear to transfer well by blotting, remains at the top of the blot, and is usually not detected by hybridization. If desired, DNA can be removed from these samples by poly(A) selection or digestion with RNase-free DNase. © 1994, Elsevier Science Publishers, B.V. All rights reserved.