RT-PCR for small rat tissue-Hes3 primer

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debdee
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RT-PCR for small rat tissue-Hes3 primer

I am trying to do RT PCR with tissue samples from subventricular zone and striatum of rat brain.
My problems now:

1. The mRNA extraction is mostly very variable was quantfied by spectro. The spectro gives different values for the same sample on repeatative estimation. How can I have approximately same amount of mRNA to make the cDNA, so that in the PCR reaction I have approx same amount of template.

2. The standard curve of one of the primers (Hes-3) in the reverse direction. It has the good amplication plot, but the standard is in the reverse direction, which makes the whole experiment to go waste. The GAPDH control standard is fine. What could be the probable problem with the primer? Has it got to do something with the primer dimer formation? but the amplification plot looks neat without much noise.

3. Is it possible to do q-PCR directly from tissue without the mRNA and cDNA synthesis, since I have a feeling that I loose a quite amount of material in the whole process.

4. One of my primers is making dimer formation (Notch). Is selecting a new primer the only solution?

Any suggestions will be highly helpful