Nucleic Acids Research, Vol 25, Issue 6 1305-1306, Copyright © 1997 by Oxford University Press
PAE Scott, K Smith, R Bicknel and AL Harris
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.
ARTICLES
A reliable external control for ribonuclease protection assays
Molecular Angiogenesis Laboratory, Imperial Cancer Research Fund, John Radcliffe Hospital, Oxford OX3 9DU, UK.
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