6.2. cDNA Synthesis

In this step, the purified mRNA-Reverse GS primer hybrids eluted from AMPure beads are extended by Reverse Transcriptase to generate cDNA (antisense strand of mRNA).

1. Prepare the Reverse Transcriptase Buffer Master Mix as follows for each sample plus 5% extra volume of all components:
    
   

   RT Buffer Master Mix Component	Volume per sample, µl
   RT-EXT Buffer, 5x	4.4
   dNTP Mix	1.1
   Water	14.3
   Hot-start RT Aptamer	1.1
   Reverse Transcriptase	1.1
   Total	22

2. Gently vortex Master Mix and spin down briefly to collect droplets. Add 22 μl of the RT Buffer Master Mix in each reaction well and resuspend AMPure beads attached to the well surface by pipetting or in the plate using an Eppendorf shaker. Briefly centrifuge the plate at low speed and place the plate in Magnetic Stand for 1 minute. Transfer 20 μl of clear supernatant without beads from each reaction well to the new plate and seal the plate.

3. Load the plate in the thermal cycler and start running the following program:
    
   

   Temperature	Time
   50°C	30 min
   72°C	10 min
   4°C	∞

*Stopping Point in procedure: cDNA samples may be stored at 4°C overnight or at –20°C for longer periods.