3Seq

3Seq

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. 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.

Pros:

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

Cons:

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

• 3SEQ: Beck AH, Weng Z, Witten DM, Zhu S, Foley JW, et al. (2010) 3′-End Sequencing for Expression Quantification (3SEQ) from Archival Tumor Samples. PLoS ONE 5(1): e8768.

1. Nussbacher J. K., Batra R., Lagier-Tourenne C. and Yeo G. W. RNA-binding proteins in neurodegeneration: Seq and you shall receive. Trends Neurosci. 2015;38:226-236

1. Guo X., Forgo E. and van de Rijn M. (2015) Molecular subtyping of leiomyosarcoma with 3' end RNA sequencing. Genom Data 5: 366-367
2. Wei G., Luo H., Sun Y., Li J., Tian L., et al. (2015) Transcriptome profiling of esophageal squamous cell carcinoma reveals a long noncoding RNA acting as a tumor suppressor. Oncotarget 6: 17065-17080
3. Nussbacher J. K., Batra R., Lagier-Tourenne C. and Yeo G. W. (2015) RNA-binding proteins in neurodegeneration: Seq and you shall receive. Trends Neurosci 38: 226-236