Despite the recent introduction of real-time PCR
methods and cDNA microarrays, competitive PCR
techniques continue to play an important role in nucleic
acid quantification because of the significantly
lower cost of equipment and consumables. In this
s...
Despite the recent introduction of real-time PCR
methods and cDNA microarrays, competitive PCR
techniques continue to play an important role in nucleic
acid quantification because of the significantly
lower cost of equipment and consumables. In this
study, we developed a construct, termed tumor suppressor-
internal standard (TS-IS) that produced polycompetitive
RNA templates as an internal standard to
quantify cellular RNA concentration of tumor suppressor
genes. This construct is composed of not only
sets of primers for detecting the expression of several
tumor suppressor genes (such as pRB, p16INK4A,
p15INK4B, p14ARF, p53, and p21WAF1), but also HPRT as
an endogenous marker. Using an internal standard
RNA that was synthesized from the TS-IS construct,
we were able to establish optimized conditions for the
quantification of tumor suppressor genes with minimal
amounts (50 ng) of cellular RNA. In addition, the
usefulness of this method was confirmed by analyzing
the expression levels of tumor suppressor genes in
fourteen hepatoma cell lines as a model. The TS-IS
assay that we used was inexpensive and a widely applicable
method that permitted the reliable and accurate
quantification of tumor suppressor genes.