FFPE Sample Prep:Odysseys in Overcycling

FFPE-derived RNA samples throw down everychallenge in the book, from degraded materials to unpredictable input amountsand transcript complexity. Traditional fixed-cycle PCR not only leads to unevenamplification, but can also trigger overcycling and burn out those fragiletranscripts. The n6 iconPCR system with AutoNorm steps in as your PCR guardian:it continuously monitors fluorescence and dynamically curtails amplification,ensuring every sample hits its sweet spot—no more, no less.​

RNA-seq Optimized, Every Well, Every Sample

Our application note demonstrates thissuperpower. Libraries amplified using standard PCR protocols (even NEB’sexcellent recommendations) risked overcycling and batch variability, especiallyacross different input levels. By contrast, libraries generated with theiconPCR and AutoNorm workflow were amplified right up to the optimal endpoint,regardless of starting RNA quantity or integrity. This not only improveduniformity across wells but consistently delivered high-quality libraries, evenfor tricky FFPE inputs.​

NEBNext Kits: Our Workflow BFF

The NEBNext kits (Ultra II Directional RNA Library Prep and rRNA Depletion)are built for robust RNA-seq performance. Combined with iconPCR precision, youcan mix-and-match different sample types and input amounts—all without endlessprotocol tweaking. The workflow yields reliable libraries even as the FFPEgremlins try their worst, sweeping away the need for input-matched batching andminimizing sample-specific fuss.​

Real-World Evidence: Why Proper Cycle Control Matters

The latest research underscores how critical PCRcycle selection and input amount are for RNA-seq data quality. Overcycling (orusing low input amounts) can drastically inflate PCR duplicates, hide expressedgenes, and muddy quantification accuracy. Platforms like iconPCR with AutoNormmake it possible to avoid these pitfalls—protecting data integrity andmaximizing the number of genes detected, as recommended by best practices.​

The n6 + NEB Combo: FFPE Challenge Crusher

So, why settle for less? At n6, we’re obsessedwith unlocking the full potential of your RNA-seq, even from the mostchallenging FFPE samples. By partnering with NEB—whose kits are the stuff oflegend—and wielding the iconPCR platform's real-time finesse, you can wavegoodbye to overamp doom and say hello to data that’s reproducible, robust, andactually fun to work with.