Enzyme Properties and Novel Activity:

  • Higher thermostability, processivity, and fidelity than retroviral reverse transcriptases, allowing full-length, end-to-end cDNA synthesis from highly structured or heavily modified RNAs.
  • Novel end-to-end template-switching activity that enables attachment of RNA-seq or PCR adapters during reverse transcription and eliminates the need for a separate RNA 3'-adapter ligation step.1 This template-switching activity greatly facilitates strand-specific RNA-seq library construction with less bias than procedures employing random hexamer primer or using RNA ligase for adapter ligation.
  • Efficient cDNA synthesis from annealed primers. The annealed primer should have a predicted Tm >60oC. Pre-incubation of enzyme with substrate in reaction mix for 30 min at room temperature and initiation of reaction by addition of dNTPs are recommended. Optimal conditions for new applications should be determined by testing a range of salt concentrations from 25 to 450 mM NaCl.
Suggested Uses for the Enzyme:

  • Comprehensive strand-specific transcriptome profiling.
  • RNA-seq of whole-cell, exosomal, plasma, and other cell-free RNAs.
  • Profiling of miRNA and other small non-coding RNAs.
  • Identification of RNA base modifications by high-throughput sequencing.
  • RIP-seq, HITS-CLIP, CRAC, ribosome profiling.
  • RNA structure mapping using methods such as SHAPE and DMS modification.
  • Synthesis of long cDNAs.
  • RT-qPCR.
  • irCLIP for characterization of RNA-protein interactions.
  • DMS-MaPseq for genome-wide or targeted RNA structure probing in vivo.
  • Analysis of FFPE tumor samples (consult our technical support)
  • Single-stranded DNA-seq via TGIRT® template-switching (consult our technical support)

Advantages of the Enzyme:

Comprehensive Transcriptome Profiling With Less Bias Than Conventional Methods.

According to a recent publication, TGIRT®-seq of ribodepleted, fragmented Universal Human Reference RNA samplesrecapitulates the relative abundance of human transcripts and spike-ins comparably to non-strand-specific TruSeq v2 and better than strand-specific Tru-Seq v3. TGIRT®-seq is significantly more strand-specific than TruSeq v3 and eliminates sampling biases from random hexamer priming that are inherent to TruSeq. TGIRT®-seq shows more uniform 5' to 3' gene coverage and identifies more splice junctions than TruSeq. TGIRT®-seq enables simultaneous profiling of mRNAs and lncRNAs in the same RNA-seq as structured small ncRNAs, including tRNAs, which are essentially absent from TruSeq datasets.

RNA-seq of whole-cell, exosomal, plasma, and other extracellular RNAs.

Fast processing time (<5 h for RNA-seq library construction through the PCR step); requires small amounts of RNA (low ng range); comprehensive transcript profiles including mRNAs and lncRNAs together with small ncRNAs, including full-length reads of tRNAs, pre-miRNAs, and other structured small ncRNAs; less bias and greater strand specificity than conventional methods.

Higher processivity and strand-displacement activity than retroviral RTs.

  • Enables construction of RNA-seq libraries of polyadenylated RNAs using an anchored oligo(dT) primer with more uniform 5' to 3' coverage than retroviral RTs without a ribodepletion step.
  • Enables RNA-structure mapping via capillary electrophoresis-based methods like SHAPE or DMS structure mapping with significantly longer read lengths and fewer premature stops than for retroviral RTs.
  • Makes it possible to obtain full-length, end-to-end cDNAs of tRNAs and other small ncRNAs, which are refractory to retroviral RTs.
RNA-seq library construction via TGIRT® template-switching in methods like miRNA profiling, RIP-seq, HITS-CLIP, CRAC, ribosome profiling.

Fast processing time (< 5 h for RNA-seq library construction through the PCR step); requires small amounts of RNA (low ng range); does not require RNA ligase, is less biased and more efficient by having fewer steps in the procedure.

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