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not reproducible : reverse - transcription PCR

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Could anyone please help me with a problem in my semi-quatitative RT-RCR? I attempt to compare the transcript level of a gene in two samplers. I got very good result last week---- sampler A has dramatically higher expression of that gene than sampler B. B-actin was used as loading control and 1:4:16 serial dilutions of both B-action and the interested gene was performed.

But when i tried to reproduce this result this week, everything goes wierd. The dramatic difference between the two samplers disappeared. And the B-actin control looks strange-- not following 1:4:16 pattern and not the same between two samplers. I have been trying repeating the experiments for a whole week but the results were always like that. I am wondering whether anyone else has similiar experience in non-reproducibility of semi-quatitative RT-PCR? Any suggestion for me to fix the problem?

I think the number of cycles in my PCR program might be one problem. I was using 40 cycles because my mRNA was extracted from only 1000 cells. I could reduce it to 35 cycles but probably cannot go any further.
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