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What are the differences between QS FFPE and QuantSeq V2 kits?

The reagents for library generation and the reaction volumes differ between QuantSeq FFPE and QuantSeq V2 protocols. Specifically:


QuantSeq FFPE

QuantSeq V2*

First strand cDNA synthesis

  • RNA input volume up to 7.5µl

  • Oligod(T) with UMIs

  • Oligo dT provided separately

  • Denaturation step 1min at 95°C

  • 10X FS buffer

  • RT reaction 15min

  • RNA input volume up to 5µl

  • No UMIs included

  • Oligod(T) primer included in the FS1 buffer

  • Skip denaturation step

  • FS1 and FS2 buffers

  • RT reaction up to 1hr

RNA removal

  • RS + RE enzyme

  • 3 µl of RS + RE master mix added per reaction

  • Additional incubation step at 37°C, prior to incubation at 95°C

  • Only RS enzyme

  • 5µl of RS added per reaction

  • Incubation at 95°C only


Second Strand Synthesis

  • 5µl SS1 buffer

  • 15 min incubation

  • 2µl SS2/E2 reaction mix

  • 10µl SS1 buffer

  • 30 min incubation

  • 5µl SS2/E2 reaction mix


  • 8µl PB

  • 20µl EB

  • 26µl PS

  • 16µl PB

  • 40µl EB

  • 56µl PS


The overall protocol time remains similar between the two library preps, with a total prep time of 4.5 hours (1.45 hours of hands-on time).

* Reaction components and volumes are provided according to the QuantSeq V2 protocol (with modifications for FFPE RNA samples).

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