Protooncogenes expression- cDNA versus RNA Probes
negar at wam.umd.edu
Thu Jun 27 12:09:21 EST 1996
To Whomever Has the Answer:
We have been following the expression of immediate-early
protooncogenes in cells stimulated with PHA. Previous findings (Reed,
et al.; Anderson, et al.), using cDNA probes, have shown that the no
detectable expression of c-fos occurs before stimulation. When these
cells are stimulated, c-fos expression increases dramtically within
15 minutes and then reapproaches baseline expression after two hours.
This represents the classical immediate-early gene response.
We are interested in studying protooncogene expression in
samples with a limited number of cells as opposed Anderson, et al.,
who were using a large number of cells from which 10-20ug of total
RNA could be obtained. With our limited sample size we then decided
to develop a quantitative RT-PCR method for detecting protooncogene
expression. The assay follows the same published procedures using an
internal standard RNA generated by in vitro transcription. The
results of our QRT-PCR methods, however, showed significant levels of
protooncogene before stimulation. More suspiciously, the stimulation
of these cells did not modulate protooncogene expression as was seen
by Reed, et al. We then tried to confirm our results by Northern Blot
analysis. Using the same in vitro transcribed RNA internal standard
that was used in the QRT-PCR, our results again showed specific
baseline expression with no modulation of protooncogene mRNA levels.
We know by sequence analysis that the antisense RNA probe is specific
to our protooncogenes.
Here is my question to you, if you choose to accept it:
1. What possible differences in the Northern Blot methods,
particularly the probes, could explain the differences seen
with our data and that of Reed, et al?
2. Since we were interested in gene expression from a limited sample
size, could the sensitivity of our RNA probe hybridization
account for the detection of basline gene expression? Also,
could this interfer with the detection of modulated gene
3. More recent Northern Blot analysis, Shlamon et al., has shown an
elevated expression of protooncogenes in the normal lung.
However, when cancerous lung tissue is assayed, decreased
protooncogene expression is observed. Does this mean that
protooncogene expression is constitutively expressed, and
does this suggest a unique tissue specific function of
Obviously, there are more questions to be asked and more expreiments
to be performed. What is your educated opinion?
Thanks for your time.
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