RNA abundance measured by a lysate RNase protection assay.
We describe a sensitive ribonuclease protection assay that we have used to measure the amount of interferon-beta RNA directly in lysates of human cells. Cell lysates were prepared in concentrated guanidine thiocyanate. Molecular hybridization with RNA probes was then performed directly in crude cell lysate, and native RNase-resistant duplexes were characterized by polyacrylamide gel electrophoresis. Comparison of interferon-beta RNA abundance by quantitative solution hybridization and lysate RNase protection showed that lysate RNase protection was highly quantitative. A high degree of reproducibility of the method was determined with a glyceraldehyde-3-phosphate dehydrogenase "housekeeping" gene probe. Sensitivity of lysate RNase protection was determined using both induced interferon-beta RNA and synthetic human endogenous reverse transcriptase RNA as target. The lysate RNase protection method was able to measure as few as 10(4)-10(5) RNA molecules.
Haines
D S
DS
Hahnemann University.
Gillespie
D H
DH
D005821:Genetic Techniques; D006801:Humans; D016899:Interferon-beta / Q000235:genetics; D012313:RNA / Q000032:analysis* / Q000138:chemical synthesis; D015347:RNA Probes; D012260:Ribonucleases / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013698:Templates, Genetic; D014407:Tumor Cells, Cultured
D015347:RNA Probes; D012313:RNA; D016899:Interferon-beta; D012260:Ribonucleases