6.2. cDNA Synthesis

In this step, the purified mRNA-Reverse GSP hybrids eluted from SPRIselect beads are extended by Reverse Transcriptase to generate cDNA (antisense strand of mRNA). The protocol below specifies volumes of reagents and Master Mixes for one pooled sample purified from one 96-well plate.

1. Prepare the Reverse Transcriptase Buffer Master Mix as follows:
    
   

   RT Buffer Master Mix Component	Volume per sample, µl
   RT-EXT Buffer, 5X	6
   dNTP Mix	1.5
   Water	19.5
   Hot-start RT Aptamer, 20X	1.5
   Reverse Transcriptase, 20X	1.5
   Total	30

2. Gently vortex Master Mix and spin down briefly to collect droplets. Add 27 µl of the RT Master Mix into pooled sample 2 ml test tube with magnetic beads (from Step 6.1) and gently resuspend SPRIselect beads attached to the tube surface by pipetting. Avoid the formation of bubbles. Briefly centrifuge the tube at low speed and place the tube in a Magnetic Stand for 1-2 minutes until the supernatant is clear. Transfer 25 μl of clear supernatant without beads from the tube to the new 0.2-0.5 ml test tube compatible with the used thermocycler.

3. Load the test tube in the thermal cycler and start running the following program (use standard 105°C heating lid temperature):
    
   

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

4. Add 1 µl of the Primer Removal Enzyme to the sample, mix contents by pipetting 3 times, spin down to collect droplets, load the sample test tube in the thermal cycler, and run the following program:
    
   

   Temperature	Time
   37°C	20 min
   95°C	5 min
   4°C	∞

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