cDNA conversion with low concentrations

Usually even reactions with 50ng/ml should work but you should be using your samples with the highest concentration that will result in the most reliable data. However, for such a low amount you should also prime with random hexamers for 10 min at 25 °C and then follow that up with 1 hour at the optimal temperature for your specific RT enzyme.

I think you need to optimize your RNA isolation first. I understand the desire to press on with what scraps you have but your reproducibility is nil if you can’t reliably extract RNA (or whatever target you are after). It’s one thing if you expect a super small yield but I would think brain tissue has plenty of RNA and you may be bumping into background noise at those concentrations. That’s a hard lesson to learn but one most of have the hard way.

Usually even reactions with 50ng/ml should work but you should be using your samples with the highest concentration that will result in the most reliable data. However, for such a low amount you should also prime with random hexamers for 10 min at 25 °C and then follow that up with 1 hour at the optimal temperature for your specific RT enzyme.