A reliable external control for ribonuclease protection assays.
A method is described for generating an external spiked human RNA control to enhance the reliability of assessment of gene expression in tumour extracts. Spiking with an external standard RNA controls for all subsequent steps of analysis on a lane by lane basis and allows for uniform comparison of the gene of interest as a fraction of total RNA, particularly when multiple samples are not available. The antisense probe that is being used to detect endogenous gene expression is also used as an external control. A sense riboprobe is made from the same vector. Because of the flanking RNA polymerase sites incorporated in both probes, hybridization with the sense riboprobe at a much lower concentration than the antisense probe generates a larger product that can be readily separated from the endogenous protected fragment. This method is generally applicable to any riboprobe that has a T3 and T7 RNA polymerase site and allows any externally added riboprobe use for assessing endogenous gene expression to be used as the external spike control.
Scott
P A
PA
Molecular Angiogenesis Laboratory, Imperial Cancer Research Fund, John Radcliffe Hospital, Oxford OX3 9DU, UK.
Smith
K
K
Bicknel
R
R
Harris
A L
AL
D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D005822:Genetic Vectors; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D009369:Neoplasms / Q000201:enzymology / Q000235:genetics; D015347:RNA Probes / Q000138:chemical synthesis; D016372:RNA, Antisense / Q000138:chemical synthesis; D012260:Ribonucleases; D014764:Viral Proteins
D015347:RNA Probes; D016372:RNA, Antisense; D014764:Viral Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D012260:Ribonucleases