Real time PCR is 99% preparation and trouble-shooting. The sample collection and tissue should be the LEAST complicated part. Designing efficient primers and creating a reliable standard curve will take you a long time. I'll need more details to give you advice on he sample collection. For now, I'll just tell "you can't rush quality".

As the protocols vary depending on the purpose of sample preparation, what is your purpose? is it for protein analysis or others assays.

Real time PCR is 99% preparation and trouble-shooting. The sample collection and tissue should be the LEAST complicated part. Designing efficient primers and creating a reliable standard curve will take you a long time. I'll need more details to give you advice on he sample collection. For now, I'll just tell "you can't rush quality".

Try waiting 3 weeks for seedlings to generate enough new leaf/shoot growth to collect 100mg of young tissue in nuclease-free 1.5ml micro-tubes (that can be frozen for later processing), before using a kit (or Dellaporta 1996) to extract nucleotides, on ice if targeting RNA. The final DNA concentration among samples should be normalized against each other, preferably by spectometry (like nanodrop) and diluted to 20ng/ul concentration before using as template. If not using fresh, be sure to avoid freeze-thaw cycles of template, especially RNA.

We can give you more specific advice if we know more about the types of samples you are collecting and what gene(s) you are testing for.

As a general bit of advice, collect more samples than you think you will need. I typically collect one full set and a second backup set (just in case). I also collect and snap freeze into liquid nitrogen.

Are you seedlings on plates or in soil? You will want to avoid any non-plant material in your samples, especially soil.