Improved Nucleic Acid QC with Parallel CE - 9

Application Note

Quality Metrics

RNA isolated from fresh tissue, it is not a definitive measure of RNA quality from FFPE samples. To solve this problem, Illumina utilized
AATI's Fragment Analyzer to develop the DV200 metric5,6, 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 construction7.

RQN

PROSize uses the RNA Quality Number (RQN) as a quality metric for total RNA samples. PROSize considers the entire
electropherogram including the 5S and Fast Region where the small RNA separates, as well as the small and large ribosomal peaks,
the baseline resolution between the peaks, the ratio of the small and large ribosomal peaks, and the degradation in front of the
small ribosomal peak when calculating the RQN. The Fragment Analyzer can easily distinguish between small RNA fragments and
degradation in the small RNA region, enabling PROSize to assign an extremely accurate RQN. The RQN is based on a scale from 1 to
10 where 1 represents completely degraded total RNA and 10 represents intact total RNA. Universal Mouse Reference Total RNA was
separated on the Fragment Analyzer with the DNF-472 High Sensitivity Total RNA Analysis Kit (Figure 2). Freshly prepared total RNA
had a ribosomal ratio of 1.9 and an RQN of 9.7, while the same sample subjected to 70oC for 10 minutes reported a ribosomal 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.

Summary
The DQN, GQN, RQN, and DV200 developed for the Fragment Analyzer and FEMTO Pulse provide reliable assessment of nucleic acid
quality and integrity. The Fragment Analyzer provides consistent quantification, sizing, and quality assessment for nucleic acids under
60 Kb, while the FEMTO Pulse is the only instrument capable of separating and sizing gDNA over 60 Kb in as little as 70 minutes. The
FEMTO Pulse also has the added benefit of extreme sensitivity, with the ability to detect a single cell amount of gDNA. Additional
background information on nucleic acid quality control, pros and cons of different types of QC analysis, and electropherogram examples
of degradation and contamination can be found on our website8.

B
RQN=5.9

RQN=9.7
28S

18S

28S/18S=1.9

Kits Used in Study:

28S/18S=0.1

*
*
18S

Written by: Chava Pocernich, Ph.D.; Steve Siembieda, M.S.
Email: techdata@aati-us.com

28S

Size (bp)

FP-1002 gDNA 165 Kb Analysis Kit
DNF-472 High Sensitivity Total RNA Analysis Kit (15nt)

Ultra-High Sensitivity Detection
Detect nucleic acid fragments and smears into the femtogram level.
Analyze the total RNA or genomic DNA from a single cell.

Automated Operation
Use of an award winning instrument design allows samples to run
continuously without human intervention, increasing laboratory
efficiency.

User-Friendly Software
Intuitive data analysis software includes functions to calculate smear
ranges, fragment flagging, customized report generation, and batch data
processing.

Size (bp)

Figure 2: Universal Mouse Reference Total RNA separated on the Fragment Analyzer with the DNF-472 High Sensitivity Total RNA Kit. (A) 0 minutes at 70o C,
RQN=9.7. (B) 10 minutes at 70o C, RQN=5.9. LM = Lower Marker.

Correlation work demonstrating that the RQN is equivalent to Agilent Technologies' RNA Integrity Number (RIN) from the Bioanalyzer
was published in 20131. Several peer reviewed publications have since stated that the RQN provided by the Fragment Analyzer
and the RIN provided by the Bioanalyzer are equivalent indicators of RNA quality2,3. Both papers utilized the Fragment Analyzer 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 Fragment Analyzer4.

5
6

https://www.illumina.com/documents/products/technotes/technote-truseq-rna-access.pdf 2014 Evaluating RNA Quality from FFPE Samples
https://www.illumina.com/content/dam/illumina-marketing/documents/products/technotes/evaluating-rna-quality-from-ffpe-samples-technical-note-470-2014-001.pdf
2016 Evaluating RNA Quality from FFPE Samples
7 https://www.illumina.com/content/dam/illumina-marketing/documents/techniques/library-qc-fragment-analyzer-app-note-770-2017-002.pdf
Scalable Nucleic Acid Quality Assessments for Illumina Next-Generation Sequencing Library Prep
8 https://www.aati-us.com/applications/nucleic-acid-analysis/

DV200 Metric

For additional application notes see our website: www.aati-us.com/application-notes

FFPE RNA samples are difficult to utilize, 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

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1 https://www.genengnews.com/gen-articles/simplifying-ht-rna-quality-quantity-analysis/4688 Simplifying HT RNA Quality & Quantity Analysis Automated CE System
Designed to Improve Rapid Assessment
2 https://www.ncbi.nlm.nih.gov/pubmed/27809311 Laser Microdissection of Tomato Fruit Cell and Tissue Types for Transcriptome Profiling
3 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5503482/ A cost-effective RNA extraction technique from animal cells and tissue using silica columns
4 https://www.aati-us.com/plant-rna-analysis/ Plant RNA Degradation Detection with the Fragment Analyzer

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Copyright ©2018 Advanced Analytical Technologies, Inc. All Rights Reserved. Reproduction, adaptation or translation without prior written permission is prohibited except as allowed under copyright laws. Fragment Analyzer™ is a trademark of Advanced Analytical
Technologies, Inc. FEMTO Pulse® is a registered trademark of Advanced Analytical Technologies, Inc. PROSize® is a registered trademark of Advanced Analytical Technologies, Inc. All production and services described herein are intended FOR RESEARCH ONLY and NOT
FOR USE IN DIAGNOSTIC PROCEDURES.

www.aati-us.com

https://www.illumina.com/documents/products/technotes/technote-truseq-rna-access.pdf https://www.illumina.com/content/dam/illumina-marketing/documents/products/technotes/evaluating-rna-quality-from-ffpe-samples-technical-note-470-2014-001.pdf https://www.illumina.com/content/dam/illumina-marketing/documents/techniques/library-qc-fragment-analyzer-app-note-770-2017-002.pdf https://www.aati-us.com/applications/nucleic-acid-analysis/ http://www.aati-us.com/application-notes https://www.genengnews.com/gen-articles/simplifying-ht-rna-quality-quantity-analysis/4688 http://www.aati-fr.com http://www.aati-us.com http://www.aati-de.com http://www.aati-uk.com https://www.ncbi.nlm.nih.gov/pubmed/27809311 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5503482/ https://www.aati-us.com/plant-rna-analysis/ http://www.aati-us.com http://www.aati-us.com

Improved Nucleic Acid QC with Parallel CE

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