A reverse time-course method for transcriptional chase analyses of mRNA half-lives in cultured cells.
Standard methods for assessing mRNA stabilities in intact cells are labor-intensive and can generate half-life (t(1/2)) measures that are both imprecise and inaccurate. We describe modifications to a conventional tetracycline-conditional transcriptional chase method for analyzing mRNA stability that significantly simplify its conduct, while generating highly reproducible and accurate t(1/2) values. The revised method-which is conducted as a reverse time course, and which accounts for interval expansion in the number of cultured cells-is validated for the analyses of mRNAs with both short and long half-lives. This approach facilitates accurate assessment of mRNA metabolism, providing a user-friendly tool for detailed investigations into their structures and functions, as well as the processes that contribute to their post-transcriptional regulation.
Department of Pediatrics Hematology, University of Pennsylvania School of Medicine and The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, United States of America. abdulmalik@email.chop.edu
Osheiza
O
Abdulmalik
Alyssa A
AA
Lombardi
J Eric
JE
Russell
D049109:Cell Proliferation / Q000187:drug effects; D004318:Doxycycline / Q000494:pharmacology; D005821:Genetic Techniques; D006207:Half-Life; D006367:HeLa Cells; D006801:Humans; D020871:RNA Stability / Q000187:drug effects / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic / Q000187:drug effects
D012333:RNA, Messenger; D004318:Doxycycline