Simplified DV200 Evaluation with theAgilent 2100 Bioanalyzer System

Technical Overview

AuthorIntroduction

Eva Graf

Quality assessment of RNA samples is crucial for the success of library preparation for
RNA sequencing workflows. The Agilent 2100 Bioanalyzer system enables the highly
reproducible evaluation of RNA sample quantity and integrity, with the Total RNA Nanoand Pico assays. In general, the RNA integrity number (RIN) is a suitable parameter toassess sample integrity for RNA sequencing. The establishment of RNA sequencing
approaches for samples originating from formalin-fixed paraffin-embedded (FFPE) tissue
created the need for further differentiation of degraded RNA samples by fragment size
distribution. The DV200 quality metric represents the percentage of RNA fragments above
200 nucleotides and shows a high correlation to the precapture library yield of FFPE
samples1.

Agilent Technologies, Inc.
Waldbronn, Germany

This Technical Overview describes the simplified evaluation of DV200 using theDV200 RNA Nano and DV200 RNA Pico assays with the Agilent 2100 Bioanalyzer
system.
Primary modifications of the DV200 RNA Nano and Pico assays are:•Predefined DV200 region•

DV200 presented in region table by default•

Result flagging according to the DV200 range•

DV200 range displayed in chip summary

Technical Details
The Agilent 2100 Bioanalyzer systemwith Agilent 2100 Expert Software
version B.02.08, the Agilent RNA 6000
Nano Kit (p/n 5067-1511) and Agilent
RNA 6000 Pico Kit (p/n 5067-1513) wereused. Unless stated, the manufacturer’s
protocols and guidelines were followed.
FFPE RNA samples were provided by
the tissue bank of the National Centerfor Tumor Diseases (NCT, Heidelberg,Germany) in accordance with theregulations of the tissue bank and theapproval of the ethics committee ofHeidelberg University.

Data Analysis
The DV200 RNA assay automaticallydefines a region from 200 to 8,000
nucleotides, as shown in Figure 1. A
corresponding DV200 is provided in theRegion Table in the % of Total column.
The RIN algorithm remains unaffected forall samples. If Results tables are selected,
DV200 can be included in exported andprinted data. In addition, the DV200 ranges
can be copied from the Chip Summarytable.

Figure 1. DV200 region of an RNA sample extracted from FFPE tissue.

Result Flagging
With the DV200 metric, degraded RNA
samples can be classified according
to their size distribution. Some RNA
sequence library protocols recommend
adapting the input amount of RNA
depending on the DV200 range of thesample1. The DV200 RNA assay flags
samples with different colors with RIN <4
for easy assessment of the DV200 range(Figure 2).

DV200 range

Color>70 %Green

50–70 %

Yellow

30–50 %

Orange<30 %RedFigure 2. Result flagging with colors representing the DV200 ranges of FFPE samples with a RIN <4.
Samples 11 and 12 are eukaryotic total RNA samples with a RIN >4, and are unaffected by result
flagging.

2Importing the Assay
The DV200 RNA Nano and Pico assays
are compatible with the 2100 Expert
Software version B.02.08 or higher. They
can be used for reanalysis of existing data
files as well as for new data acquisition.
To use the DV200 RNA Nano or Picoassay with the 2100 Expert Software,
download it from the Agilent website:www.genomics.agilent.com. Store theDV200 RNA Nano.xsy or DV200 RNA
Pico.xsy file in the RNA assay folder(C:\Program Files (x86)\Agilent\2100
bioanalyzer\2100expert\assays\RNA).
To apply the DV200 on an existing data file:1.

Open the file with the 2100 Expert
Software.

2.

Navigate to the Assay Properties
tab.

3.

Click Import Setpoints and select thecorresponding. xsy file (Figure 3).

Figure 3. Importing the DV200 RNA assay into existing data files.

The DV200 RNA Nano assay can only be
used to reanalyze Eukaryote Total RNA
Nano data files, and the DV200 RNA Picoassay is only for Eukaryote Total RNA
Pico data files.
Assay setpoints and result flagging will
be applied to the data file and can besaved afterwards.
To run samples with the DV200 RNA
Nano and DV200 RNA Pico assay:
1.

Prepare the samples and the RNA
chip according to the RNA Nano or
Pico Kit Guide.

2.

When the chip is prepared andplaced in the 2100 Bioanalyzer
instrument, go to the instrument
context, click Assay Selection,and choose the corresponding
DV200 RNA assay. The assay will bepart of the general assay selection
when it has been stored in the RNA
assay folder (Figure 4).

3.

Figure 4. Selecting the DV200 RNA assay to run an RNA chip.

To start the run, click Start.

3ConclusionThe DV200 RNA Nano and DV200
RNA Pico assays are useful tools toautomatically define DV200 regions for
degraded RNA samples extracted fromFFPE tissue. Once the assay is imported,
it can be used to run the Agilent 2100
Bioanalyzer RNA chips, or to reanalyze
existing data files. The DV200 results canbe saved, exported, and displayed inreports.

References
1. Evaluating RNA Quality from FFPE
Samples. Illumina, Technical Note,publication number 470‑2014 001.
https://www.illumina.com/
documents/products/technotes/
technote-truseq-rna-access.pdf

www.agilent.com/genomics/bioanalyzer
For Research Use Only.
Not for use in diagnostic procedures.
This information is subject to change without notice.
© Agilent Technologies, Inc., 2017
Published in the USA, August 1, 20175991-8287EN