Isolation of biologically active RNA from human autopsy for the study of cystic fibrosis.
Biologically active RNA has been isolated from several tissues implicated in the pathogenesis of the genetic disease cystic fibrosis. To ensure that mRNA from stomach and pancreas has not been degraded, it must be isolated within 2 h postmortem, while intact RNA can be isolated from lung material up to 20 h postmortem. It is imperative that tissue removed during autopsy be dispersed in a strong denaturant buffer (guanidine isothiocyanate) to inactivate nucleases. While stomach and pancreas yields of RNA are low, relative to the amount of gland material, yields of RNA from lung samples are sufficient for the further isolation of mRNA by oligo(dT)-cellulose chromatography. The biological integrity of subsequently isolated mRNA has been assessed by AMV-reverse transcriptase cDNA synthesis as well as by in vitro translation. Protein products generated in this manner show distinct differences from mRNA translation patterns of age- and sex-matched controls when compared with mRNA from CF samples. R0t analysis of cDNA libraries thus generated indicates that the complexity of such libraries is representative of the starting RNA species.
D001344:Autopsy; D003550:Cystic Fibrosis / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000032:analysis; D006801:Humans; D008168:Lung / Q000032:analysis; D009693:Nucleic Acid Hybridization; D011180:Postmortem Changes; D012333:RNA, Messenger / Q000302:isolation & purification*; D013048:Specimen Handling
D012333:RNA, Messenger; D004247:DNA