Workflow FAQ for Illumina Rapid RNA Kits

What is the key workflow difference of Rapid RNA kits?

Proprietary chemistry allows cDNA synthesis and adapter tagging in a single step.

Is the cDNA generated double-stranded?

Yes, the cDNA generated is double-stranded. Tagmentation can only occur on double-stranded DNA.

Do Rapid kits generate stranded data in the final library?

No, Rapid RNA kits generate non-stranded data. For stranded RNA data, use either Illumina Stranded mRNA or Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus or Ribo-Zero Plus Microbiome.

Note that the earlier-generation Illumina RNA Prep with Enrichment kit has always generated non-stranded data as well; this remains non-stranded with the Rapid version, as well.

What is the library backbone of Rapid RNA libraries? How does the introduction of indexes differ?

Illumina Rapid RNA libraries have a "Nextera backbone" due to tagmentation with the EBLTS reagent and the use of Illumina DNA/RNA UD Indexes. In Illumina Rapid RNA workflow, indexes are added in a single step. This is the same mechanism of index addition as the earlier Illumina RNA Prep with Enrichment (IRPE) kit.

In contrast, in Illumina Stranded mRNA and Stranded Total RNA workflows, index adapters are added in two steps: first ligation of index anchors (without UMIs), followed by PCR with index adapters. See the article Understanding the differences between Illumina index chemistries and anchor function, for more information.

Rapid RNA Library structure:

Can users expect normalized libraries output from the Rapid RNA prep as long as it falls within the recommended input range (eg 10-100 ng)? At what concentration do the EBLTS saturate so they produce normalized libraries, if not?

No, saturation is not expected with the recommended input amounts. Always quantify and manually normalize individual samples at the end of library prep.

For any feedback or questions regarding this article (Illumina Knowledge Article #10213), contact Illumina Technical Support [e-mail].