Protooncogenes expression- cDNA versus RNA Probes

negar moshiri 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 
	expresion?
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 
	protooncogene expression. 

Obviously, there are more questions to be asked and more expreiments 
to be performed. What is your educated opinion?

Thanks for your time.

Sincerely, 

Ernest Brown



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