cDNA Generation

Rather than doing a combined RT-PCR, I use a protocol that generates the cDNA first. 

Typically, I will prepare cDNA from 1ug of RNA.  Sometimes, animal tissue doesn't provide that much RNA.  200ng is acceptable.  If your concentrations won't allow you to use 1ug, use the most you can. 

You can use either oligo dT (which converts only mRNA) or random hexamers (which converts mRNA and rRNA).  I prefer random hexamers, but either is OK. 

A real-time calculator is on the main page that will let you plug in your values and get a formula for running the cDNA generation.