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Next Generation Sequencing: Total RNA

The composition of RNA in a cell can be roughly estimated at 80% ribosomal RNA, 15% transfer RNA, and 5% messenger RNA. With total RNA sequencing, all of those subtypes (as well as any small RNAs) are converted into cDNA for sequencing.

How it Works:

We use the Ovation(R) RNA-Seq v2 kit from NuGEN for conversion of total RNA to cDNA, which uses random hexamer priming (along with 3' poly(A) priming) for first strand synthesis, and an isothermal strand-displacement process (SPIA) for unbiased cDNA amplification.

After cDNA is generated and amplified, it is fragmented and ligated to Illumina adapters in the same way as a genomic DNA library would be built.

Pros:

  • Extremely low risk of bias
  • Good results even from degraded RNA
  • Can be done with very low amounts of RNA (>500pg)

Cons:

  • More sequencing reads are needed (since 80% will be reading rRNA), increasing cost
  • Optimized for mammalian systems, so may have issues with other source organisms

Recommendations:

  • Opt for total RNA sequencing if your RNA has a RIN score of less than 6 or you have less than 100ng of total RNA.