3′-End Sequencing for Expression Quantification

3′-end Sequencing for Expression Quantification (3Seq) is able to isolate highly degraded mRNA from formalin-fixed paraffin-embedded (FFPE) tissue samples for quantitative genome-wide expression analysis (Beck et al., 2010). First, mRNA is isolated from total RNA by poly(A) selection. Next, an oligo(dT)-P7 RT primer is annealed to the 3′ end of the mRNA fragment to synthesize the first cDNA strand via reverse transcription (RT). A second cDNA strand is generated from the first strand, and P5 adapters are ligated on the opposite end from the P7 adapters. The fragments containing the adapters are amplified by PCR and are ready for sequencing. 3Seq can also be used for fresh-frozen tissue samples.


  • Sequences highly degraded mRNA from FFPE or fresh-frozen tissue samples


  • High internal priming rate
  • Homopolymeric nature of poly(A) tail often causes polymerase slippage


Illumina Library prep and Array Kit Selector


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