Agilent eBook - 21

Best Practices with an Agilent Parallel CE System

ratio of 0.1 and an RQN of 5.9. A large RQN value
indicates higher quality RNA with minimal degradation
in the sample. The concentration, RQN, and ribosomal
ratio are automatically reported in ProSize, allowing for
easy evaluation of total RNA quality.
Correlation work demonstrating that the RQN is
equivalent to the RNA integrity number (RIN) from
the Bioanalyzer was published in 20133. Several peer
reviewed publications have since stated that the RQN
provided by the 5200 Fragment Analyzer system and
the RIN provided by the Agilent 2100 Bioanalyzer
system are equivalent indicators of RNA quality4,5.
Both papers used the 5200 Fragment Analyzer system
for assessing RNA quality and presented several
electropherograms as examples. Application of the
RQN can be found in the Application Note: Plant RNA
Degradation Detection with the Agilent 5200 Fragment
Analyzer System6.
DV200 metric

FFPE RNA samples are difficult to use, as degradation
due to fixation and storage conditions is often quite
extensive. It is important to evaluate the quality of
each FFPE RNA sample before proceeding with library
preparation to eliminate highly degraded samples
containing RNA fragments smaller than the optimal
size range. Although RQN values are reliable metrics
for evaluating the quality of RNA isolated from fresh
tissue, it is not a definitive measure of RNA quality

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from FFPE samples. To solve this problem, Illumina
used the Fragment Analyzer system to develop the
DV200 metric7, which calculates the percentage of
RNA fragments greater than 200 nucleotides in size.
The DV200 metric is used to determine the minimal
RNA input required for successful library preparation
and reproducible results. Given the strong correlation
between DV200 values and library yield, the DV200 metric
is ideal for assessing FFPE RNA quality prior to library
construction8.
Conclusion
The DQN, GQN, RQN, and DV200 developed for the 5200
Fragment Analyzer system and Femto Pulse system
provide reliable assessment of nucleic acid quality and
integrity. The 5200 Fragment Analyzer system provides
consistent quantification, sizing, and quality assessment for nucleic acids under 60 kb, while the Femto
Pulse system is the only instrument capable of separating and sizing gDNA over 60 kb in as little as
70 minutes. The Femto Pulse system also has the
added benefit of extreme sensitivity, with the ability
to detect a single cell amount of gDNA. n

References
1. Pocernich, C.; Uthe, J.; Wong, K-S. Genomic DNA Sizing
and Quality Control on the Agilent Femto Pulse System,
Agilent Technologies Application Note, publication number 59940516EN, 2017.
2. Evaluating DNA Quality from FFPE Samples. Guidelines for
Obtaining High-Quality DNA Sequencing Results from FFPE
Samples Using the TruSeq Exome Library Preparation Kit.
Illumina Technical Note, publication number 770-2015-035, 2016.
3. Wong, K-S; Pang, H. Simplifying HT RNA Quality and Quantity
Analysis: Automated CE System Designed to Improve Rapid
Assessment. Genet. Eng. Biotechn. N. 2013, 33(2).
4. Martin, L. B.; Nicolas, P.; Matas, A. J.; Shinozaki, Y.; Catalá, C.;
Rose, J. K. Laser Microdissection of Tomato Fruit Cell and Tissue
Types for Transcriptome Profiling. Nat. Protoc. 2016, 11(12),
2376-2388.
5. Escobar, M. D.; Hunt, J. L. A Cost-Effective RNA Extraction
Technique from Animal Cells and Tissue Using Silica Columns. J.
Biol. Methods 2017, 4(2).
6. Pocernich, C.; Warzak, D.; Wong, K-S. Plant RNA Degradation
Detection with the Agilent 5200 Fragment Analyzer system.
Agilent Technologies Application Note, publication number
5994-0518EN, 2017.
7. Evaluating RNA Quality from FFPE Samples. Guidelines for
Obtaining High-Quality RNA Sequencing Results from Degraded
RNA with Illumina RNA Enrichment Assays. Illumina Technical
Note, publication number 470-2014-001, 2016.
8. Scalable Nucleic Acid Quality Assessments for Illumina
Next-Generation Sequencing Library Prep. Simultaneous
Qualification and Quantification of Nucleic Acids with the
Fragment Analyzer, Illumina Technical Note, publication number
770-2017-002-A, 2017.

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For Research Use Only. Not for use in diagnostic procedures.
This information is subject to change without notice.
© Agilent Technologies, Inc. 2019
5994-0521EN

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