ExactSTART™ Eukaryotic mRNA 5´- & 3´-RACE Kit

Transcriptome Analysis: ExactSTART™ Tools for Rapid and Efficient cDNA Preparation for Deep Sequencing, Cloning,and Expression Profiling (940K PDF poster)

Applications

  • Discover alternative transcription and polyadenylation sites of mRNAs.
  • Unambiguously map the exact 5´ and 3´-ends of mRNAs.
  • Identify and characterize splice variants and transcript homologs.
  • Produce double-strand cDNA from eukaryotic mRNA for RACE or other RNA transcript discovery method.

One common RACE template preparation procedure relies on the addition of non-template-coded dNTPs to the 3´-end of the cDNA produced during reverse transcription of the RNA. Such deoxynucleotide additions make it difficult to identify the true 5´-end of the transcript. In addition, dNTPs are added to some cDNA molecules more efficiently than others which may result in loss of transcripts from the final population of RACE-amplified cDNA.

In contrast, the ExactSTART Eukaryotic mRNA 5´ & 3´-RACE Kit procedure uses a rapid and improved "oligo-tagging" process to tag the exact 5´-nucleotide of mRNAs and then produces 5´ and 3´-tagged cDNA that can be used as is, converted to double-strand cDNA, or amplified by PCR to produce enriched (amplified) ds cDNA for 5´ and 3´-RACE or other application such as production of next-gen sequencing template.

The ExactSTART™ Eukaryotic mRNA 5´ & 3´ RACE Kit produces 5´ and 3´-end tagged cDNA or amplified double-strand cDNA (ds cDNA) from 3´-polyadenylated RNAs (e.g., eukaryotic mRNA) that have:

5´-Cap / 3´-polyadenylated ends   5´ m7GpppN-------[A]n 3´  or
5´-monophosphorylated / 3´-polyadenylated ends   5´ pN----------------[A]n 3´  or
5´-triphosphorylated / 3´-polyadenylated ends   5´ pppN-------------[A]n 3´

Benefits

  • Accurate – Tag the exact 5´-nucleotide of mRNAs to unambiguously map the true transcription site of an mRNA. Unlike other 5´-RACE template production methods, the ExactSTART approach does not add template-independent nucleotides during the tagging process.
  • Fast – Generate RACE template in less than a day.
  • Easy – Simplified protocol requires no gel purification steps.
  • Flexible – Reactions generate cDNA, ds cDNA or amplified ds cDNA for use as RACE template or other application such as production of Next-gen sequencing template.

Figure 1 (click to enlarge). Schematic overview of the process for the ExactSTART™ Eukaryotic mRNA 5´- & 3´-RACE Kit.