1. Mix and heat the RNA Template, 2X Reaction Mix, and Primers to 65 °C for 5 minutes. Allow the RNA Template to stand in an ice bath for at least 1 minute.
2. Add the following components to a PCR tube on ice: 
   

   Component	Volume
   RNA Template	Variable 10 pg – 2 µg total RNA or 10 pg – 500 ng mRNA)
   2X Reaction Mix	10 µl
   Primers	1 µl
   Enzyme Mix	2 µl (200 U)
   RNase Inhibitor	1 µl (20-40 U)
   Water	Variable, up to 20 µl
   Total Volume	20 µl

    

3. Mix by gently pipetting up and down.
4. Close the lid on the tube and incubate in a temperature-controlled water bath at 55 °C for 50 minutes for the extension step.
   Note: the optimal temperature for extension is likely between 42-60 °C.
5. Incubate the tube at 70 °C for 15 minutes to inactivate the Reverse Transcriptase before amplification.

Notes:

• Avoid cross-contaminating the RNA template (total RNA, synthetic RNA transcript, poly(A) + mRNA) with DNA.
• Recommended primers (per 20 µl reaction):
  • 2.5 µM of oligo(dT) – anneal to 3′-poly(A) + mRNA
  • 2.5 ng/µl of random primers – anneal at non-specific sites of RNA templates
  • 2 µM of gene-specific primers