RT smears troubleshooting

A quick look at my history of RT success to ID what started going wrong?


  • not much smearing in early samples. Incorporation rate increases with total amount of mRNA added.
  • Using beads to concentrate mRNA increases production.
  • Using pure mRNA T7 further increases production
  • Small samples have worse smears just after the high quality maxima integration (8 probes at 800 pmol P1, and 1 at nmol P2, 2 at 600 and 800 pmol P2).

The decline

  • Correlates with switch to new P1. P3 was doing better than P1 for a little while after the switch.
  • correlates with switch to murine RNase inhibitor from NEB (cheaper). Best runs were with RNasin Plus.
  • This is interesting, RNasin plus advertises thermal stability up to 70C. Maybe this is the trick — NEB’s inhibitor says it should be used below 50C.
  • correlates with lower IVT yields
  • Same protocol essentially. Had higher maxima when we got high incorporation, but bringing maxima back up to high concentration doesn’t restore high incorporation.
  • Nothing else changed really in protocol.



  • some contamination of reagents.
  • switch of RNase inhibitor.
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