Illumina Workflow: Pippin for RNA Studies

Illumina sequencers are far and away the most popular platform on the market. Since so many Sage customers use their Pippins with these instruments, we’re taking a look at some of the most common or interesting applications of automated DNA sizing with the HiSeq, MiSeq, and even the GA II or GA IIx workhorses. If you missed it, check out our recent blog about using Pippin for Illumina mate-pair sequencing. Our focus today is small RNAs.

Customers like Kevin Knudtson from the University of Iowa and Stuart Levine at MIT say that Pippin Prep is an integral part of the miRNA pipeline. Knudtson, who runs the university’s DNA Facility, says his team will not even “consider a manual gel extraction for microRNAs.” In their hands, Pippin is remarkably effective at isolating miRNAs, even with a lot of adapter-dimers, primers, and other small content in the neighborhood.

According to Levine, director of MIT’s BioMicro Center, “The high percentage gels on the Pippin allow us to cut out bands of the right size for microRNAs.” In a core facility, the alternative — manually cutting bands from a gel — is not economically feasible. His team also uses Pippin Prep for splice variant analysis with an RNA-seq workflow. “Some of the RNA-seq methodologies, when you’re doing de novo sequencing of transcriptomes and want to do assemblies, tend to perform better when the size distribution of the library inserts is very tight,” he says.

We also worked with New England Biolabs as they developed their NEBNext Multiplex Small RNA Kit, which functions best with Pippin sizing. A recent paper from scientists at the Mayo Clinic, Institute for Systems Biology, and University of Illinois demonstrated this workflow in a project examining the effects of vitamin D on microRNA regulation of gene expression in zebrafish. They enriched for miRNAs of interest with Pippin and the NEBNext kit. To learn more about how these products fit together, check out this app note.

Our blog series will continue with peeks into how scientists are using Pippin and Illumina sequencers together for ChIP-seq, ddRADseq, and more. Check back soon for more!

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