Protocol for Use with Previously Fragmented RNA - NEBNext mRNA First Strand Synthesis Module (E7525)

Starting Material: 50–250 ng of purified mRNA fragmented to 200 nt and cleaned up in 13.5μl of nuclease free water

First Strand cDNA Synthesis

  1. Mix the following components in a sterile PCR tube:
    Fragmented mRNA   13.5 μl
    Random Primers   1 μl
    -------------------------------------------------------------------------
    Total volume   14.5 μl
  2. Incubate in a preheated thermocycler for 5 minutes at 65°C.
  3. Spin tube briefly and place on ice.
  4. To the fragmented mRNA and Random Primers add:
    5X First Strand Synthesis Reaction Buffer   4 μl
    Murine RNase Inhibitor   0.5 μl
    -------------------------------------------------------------------------
    Total volume   19 μl
  5. Incubate in a preheated thermocycler for 2 minutes at 25°C.
  6. Add 1 μl ProtoScript II Reverse Transcriptase to the reaction.
  7. Incubate sample as follows:
    10 minutes at 25°C
    50 minutes at 42°C
    15 minutes at 70°C
    Hold at 4°C 
  8. Place the tube on ice.
  9. Proceed directly to second strand synthesis using NEBNext mRNA Second Strand Synthesis Module (NEB #E6111).