A reliable external control for ribonuclease protection assays
A reliable external control for ribonuclease protection assays
Prudence A. E.
Scott
,
K.
Smith
,
Roy
Bicknel
and
A. L.
Harris*
Molecular Angiogenesis Laboratory, Imperial Cancer Research Fund, John Radcliffe
Hospital,
Oxford
OX3 9DU,
UK
Received January 2, 1997;
Accepted February 3, 1997
ABSTRACT
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.
Ribonuclease protection assays are commonly used to assess quantitative
expression of genes in human cancer and relate tumour biology to gene
expression. To standardise for unequal sample extraction, measuring levels of
an internal control gene co-expressed in the sample offers a means of ensuring that equal amounts of
each sample are recovered, as well as equal amounts are loaded and detected
during the subsequent biochemical analysis. Variation in the level of the
internal control may be due to poor extraction or losses within the experiment
after the initial aliquotting of the RNA. This requires that the internal
control gene be expressed at a constant level under all conditions, and one
used frequently is glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (
1
-
5
). Several problems exist in the use of GAPDH as an internal control,
particularly for measuring gene expression in RNA extracted from tumours. GAPDH
is known to be regulated in a biphasic manner during the cell cycle where 19-fold variations in mRNA have been observed (
6
). It is regulated by a number of factors including cellular proliferation (
7
), hypoxia (
8
), dihydroxyvitamin D in breast carcinoma cells (
9
) and by hormones such as dexamethasone
in vivo
(
10
).
An external control could be used as an additional way to assess gene expression
quantiatively. Spiking with an external standard mRNA after extraction would
control for all subsequent steps of analysis on a lane-by-lane basis. Thus, samples with low internal standard gene expression
could be due to low expression in the tumour, degradation during extraction or
losses in the experiment. Use of the spike control would show if low expression
is due to a step subsequent to extraction. The RNA loading is already
standardised by spectrophotometry and ethidium bromide staining of RNA samples
run on gels first. If the level of the spike control was uniform and the
internal control was low this would suggest endogenous variation of the
internal control mRNA. Since multiple samples are not available for repeated
extractions and comparisons in primary tumours or many human samples, the use
of the spike control allows for a uniform comparison of expression of the gene
of interest as a fraction of total RNA.
In order to use both the endogenous control gene and the external spike control
with the minimum number of different probes we have developed a method that
utilises the existence of two RNA polymerases within most vectors. When an
antisense riboprobe is transcribed from the DNA construct, in addition to the
insert that is transcribed, the vector sequence between the RNA polymerase and
the start of the insert is also transcribed. Most common vectors have two RNA
polymerases flanking either side of the polycloning site-T7 and T3 RNA polymerases in the case of Bluescript. The first strand of,
for example, the GAPDH riboprobe is generated by linearising the
Bluescript/GAPDH construct at the 5' end (or amino-terminus) of the insert with
Hin
dIII and transcribing from the opposite end with the T3 RNA polymerase to form
an antisense strand for probing endogenous mRNA (a protected fragment size of
120 bases). By linearising the 3' end of the insert and using the T7 RNA polymerase, a complementary sense
strand is formed which hybridises to the first probe generated, and also
competes with endogenous GAPDH. If the second riboprobe is designed, not to cut
immediately after the insert but several bases further downstream, the
additional vector sequence transcribed overlaps with, and is complementary to
the first probe generated and thus will also hybridise (Fig.
1
). The protected fragment (150 bases) from such a hybridisation satisfies the
two main requirements: that it is larger than the endogenous protected
fragment, thereby providing a distinct band for quantification (while also
assaying and demonstrating variations in the endogenous GAPDH expression at the
same time).
REFERENCES
1 Pimentel, J. L. J., Wang, S. and Martinez, M. M. (1994) Kidney Int., 45, 1614-1621.
2 Roach, T. I. A., Chatterjee, D. and Blackwell, J. M. (1994) Infect. Immun., 62, 1176-1184.
3 Watanabe, J., Hayashi, S. I. and Kawajiri, K. (1994) J. Biochem. (Tokyo), 116, 321-326.
4 Ihl, V. R., Marquetant, R., Bremerich, J. and Strasser, R. H. (1995) J. Mol. Cell. Cardiol., 27, 437-452.
