3Seq
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.
Advantages:
- Sequences highly degraded mRNA from FFPE or fresh-frozen tissue samples
Disadvantages:
- High internal priming rate
- Homopolymeric nature of poly(A) tail often causes polymerase slippage
Reagents:
Illumina Library prep and Array Kit Selector
Reviews:
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
References:
Riemondy K., Wang X. J., Torchia E. C., Roop D. R. and Yi R. MicroRNA-203 represses selection and expansion of oncogenic Hras transformed tumor initiating cells. Elife. 2015;4:
Wei G., Luo H., Sun Y., et al. Transcriptome profiling of esophageal squamous cell carcinoma reveals a long noncoding RNA acting as a tumor suppressor. Oncotarget. 2015;6:17065-17080
Guo X., Forgo E. and van de Rijn M. Molecular subtyping of leiomyosarcoma with 3′ end RNA sequencing. Genom Data. 2015;5:366-367
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History: 3Seq
Revision by sbrumpton on 2017-06-21 07:50:23 - Show/Hide
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.
Advantages:- Sequences highly degraded mRNA from FFPE or fresh-frozen tissue samples
Disadvantages:- High internal priming rate
- Homopolymeric nature of poly(A) tail often causes polymerase slippage
Reagents:Illumina Library prep and Array Kit SelectorReviews: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;38:226-236References:Riemondy K., Wang X. J., Torchia E. C., Roop D. R. and Yi R. MicroRNA-203 represses selection and expansion of oncogenic Hras transformed tumor initiating cells. Elife. 2015;4:Wei G., Luo H., Sun Y., et al. Transcriptome profiling of esophageal squamous cell carcinoma reveals a long noncoding RNA acting as a tumor suppressor. Oncotarget. 2015;6:17065-17080Guo X., Forgo E. and van de Rijn M. Molecular subtyping of leiomyosarcoma with 3' end RNA sequencing. Genom Data. 2015;5:366-367