1. RNA extract in T₁₀E₁8.5/EtOH stored at -80^oC Biofreezer.
2. TermoScript reverse transcriptase Kit including Synthesis Buffer (Invitrogen)
3. Poly(A) or Random pd(N)₆ primer (Invitrogen or AP Biotech 272166 dissolved in T₁₀ buffer at 250ug/ml )
4. RNase inhibitor (Promega: RNasin, Cat# N251A)
5. RNase-free H₂O
6. RNase H
7. RNase-free Eppendorf tubes
8. Pipettes and tips
9. Centrifuge (>3,000 xg)
10. Vortexer
11. SpeedVac
12. ThermoMixer or Heating block
13. Ice

1. Thaw RNA stock and put on ice. Vortex and take the volume equivalent to 10 ug of RNA and put in 1.5 ml RNase-free tube.
2. Spin at 14000 rpm for 15 min at 4^oC. Aspirate liquid completely. Wash pellet with the same volume of 80% EtOH as RNA stock, aspirate completely and vacuum dry in SpeedVac for 5 min without heating.
3. Dissolve pellet in 20ul RNase-free water.
4. Heat at 90^oC on ThermoMixer for 5min and put on ice immediately.
5. Add 2ul RNasin to protect RNA from degradation.
6. Make cocktail solution as in the table below, DO NOT add reverse transcriptase, yet. (column RT+, RT- is only for demonstration only at this step)

7. Split the mixture 19ul x 2 into two 1.5 ml tubes. Add 1ul reverse transcriptase to one of them (mark as RT+) and 1ul H2O to another tube as control (RT-).
   Mix gently and incubate at 50 ^oC for 45-60 min (for random-hexamer primed, incubate at RT for 10min first).
8. Inactivate at 95^oC for 5 min, chill on ice.
9. To remove RNA complementary to the cDNA, add 1ul RNase H and incubate at 37^oC for 20min.
10. Apply 2ul of the reaction for 50ul PCR (run 40 cycles for analysis and 25 cycles for preparation).

For some brand of DNA polymerase like Pfu DNA polymerase, the RT reaction needs to be cleaned by phenol/CIA- isopropanol precipitation or other method before PCR. So if the PCR doen't work, clean the RT reaction and try again. For some brand like Taq or Tgo DNA polymerases, the cleaning step is not neccessory.