FFPE RNA Quality Assessment with the Agilent Bioanalyzer and TapeStation Systems

Importance of the Topic

Formalin fixation followed by paraffin embedding (FFPE) is widely used to preserve clinical and research tissue specimens, maintaining cellular morphology over long periods. However, formalin chemistry induces fragmentation and chemical modifications of RNA, complicating downstream applications such as next-generation sequencing (NGS). Reliable assessment of RNA integrity in FFPE samples is therefore critical for ensuring data quality, conserving precious samples, and optimizing sequencing workflows.

Study Objectives and Overview

This overview compares the performance of two Agilent automated electrophoresis platforms—the 2100 Bioanalyzer and the 4200 TapeStation—in evaluating FFPE-derived RNA integrity. Multiple tissue sources (cow liver, cow cerebellum, mouse liver, pig spleen) were assayed using four kits/assays to determine consistency in RNA Integrity Number (RIN/RINe) and the DV200 metric, which quantifies the percentage of RNA fragments above 200 nucleotides.

Methods and Instrumentation

Four FFPE RNA samples were extracted and diluted to concentrations suitable for each system. The Bioanalyzer employed the RNA 6000 Nano and RNA 6000 Pico kits, while the TapeStation used the RNA ScreenTape and High Sensitivity ScreenTape assays. Each sample was run in triplicate without heat denaturing, following manufacturer guidelines. Electropherograms were analyzed with Bioanalyzer Expert software (DV200 assay configuration) and TapeStation software using the region mode to calculate the DV200 percentage.

Main Results and Discussion

Electropherogram profiles from both platforms exhibited similar fragmentation patterns, with a peak of small RNA fragments below 200 nt and a smear extending through higher sizes. For mouse liver RNA, the Bioanalyzer reported a DV200 of 58.0% versus 62.3% on the TapeStation, indicating comparable assessments. Across all samples, average DV200 values between systems differed by less than 5%, and percent coefficients of variation (%CV) remained below 6% for all replicates. Within each instrument, the two kit/assay options yielded consistent DV200 results, demonstrating reliable performance irrespective of concentration range or sensitivity level.

Advantages and Practical Applications

• Provides quantitative QC to predict NGS library yield from degraded FFPE RNA.
• Enables researchers to screen samples before committing to expensive sequencing runs.
• Offers flexibility through interchangeable kits/assays on both Bioanalyzer and TapeStation.
• Supports standardization of FFPE RNA workflows across labs and studies.

Future Trends and Opportunities

Advancements may include integrated software algorithms for automated fragment sizing, machine learning models to correlate DV200 with sequencing success, and expanded assay chemistries for ultra-low-input samples. Emerging platforms might combine electrophoretic QC with multiplexed fragment analysis or digital microfluidic solutions, further streamlining FFPE RNA evaluation in diagnostic and research settings.

Conclusion

Both the Agilent 2100 Bioanalyzer and the 4200 TapeStation deliver robust, reproducible DV200 and RIN/RINe metrics for FFPE RNA quality assessment. Their comparable performance and interchangeable assays empower users to confidently evaluate sample suitability for NGS applications, optimizing resource use and data reliability.

Reference

1. Agilent Technologies. DV200 Evaluation with RNA ScreenTape Assays. Technical Overview, Publication 5991-8355EN, 2017.
2. Agilent Technologies. Simplified DV200 Evaluation with the Agilent 2100 Bioanalyzer System. Technical Overview, Publication 5991-8287EN, 2017.
3. Illumina. Evaluating RNA Quality from FFPE Samples. Technical Note, Publication 470-2014-001, 2016.