Technical Overview

DV200 Evaluation withRNA ScreenTape Assays

AuthorAbstract

Eva Graf
Agilent Technologies, Inc.
Waldbronn, Germany

The success of library preparation for RNA sequencing workflows depends highlyon the quality of RNA starting material. The RNA integrity number equivalent (RINe)obtained with Agilent RNA ScreenTape assays is a reliable and reproducible metricto evaluate sample integrity for standard RNA sequencing. Library preparation ofRNA samples originating from formalin‑fixed paraffin‑embedded (FFPE) tissue ischallenging, due to RNA degradation and restricted sample volume, thus tailored
protocols are recommended for FFPE RNA library preparation. The fragment size
distribution of FFPE RNA samples has a major influence on the library yield, andcan be represented as the percentage of RNA fragments above 200 nucleotides by
the DV200 quality metric1. The Agilent RNA ScreenTape and Agilent High Sensitivity
RNA ScreenTape assays are convenient for highly reproducible DV200 evaluationof degraded RNA samples extracted from FFPE tissue. The Agilent TapeStation
Analysis software enables simple assessment and processing of DV200 data byregion analysis, which can be automated for repeated DV200 analysis.

Technical Details

Data Analysis

The Agilent 4200 TapeStation systemwith Agilent TapeStation Analysis
software version A.02.02 was used.
RNA ScreenTape (p/n 5067-5576)
with reagents (p/n 5067-5577 and5067‑5578) and Agilent High Sensitivity
RNA ScreenTape (p/n 5067-5579)
with reagents (p/n 5067-5580 and5067-5581) were obtained from
Agilent Technologies. Unless stated,the manufacturer’s protocols andguidelines were followed. All samples
were analyzed with a ladder run on each
ScreenTape. RNA samples extracted
from FFPE tissue were kindly provided
by the tissue bank of the National Center
for Tumor Diseases (NCT, Heidelberg,Germany) in accordance with theregulations of the tissue bank, and theapproval of the ethics committee ofHeidelberg University.

The DV200 represents the percentageof RNA fragments larger than
200 nucleotides with respect to all
RNA fragments. TapeStation Analysis
software can be used to calculate theDV200 by defining a region for fragments
larger than 200 nt. The software provides
several ways for region setup, suitable
for low to high sample throughput.
Consult the Agilent Information Center(AIC) for further information and how-to
videos about region analysis.

Region Setup for Data Files
To define a DV200 region as default for
all samples in an individual data file,click Region, then click Region Settings,
and enter the lower and upper
limits of the region in the dialog box(Figure 1). To identify the region in theelectropherogram, type DV200 as regioncomment. To apply the region to all
samples in the active file, select This File.

Region Setup for Individual Samples
To define a DV200 region for individual
samples, click Region, then right click inthe electropherogram to add a region.
The region borders can be individually
adjusted by shifting to the left or to theright.

1. Click Region

2. Click Region Settings

3. Click, and enterregion borders

4. Apply to the active file,or to all files

Figure 1. Setting up default regions to calculate DV200 with Agilent TapeStation Analysis software.

2

Automated Region Setup
To define a DV200 region as default forreoccurring FFPE RNA sample analysis,click Region, then click Region Settings,
and enter the lower and upperlimits of the region in the dialog box(Figure 1). To identify the region in theelectropherogram, type DV200 as regioncomment. To apply the DV200 region to all
files of the same assay as the active file,select Assay Files.

When DV200 regions are defined, thevalue is provided in the region tablein the % of Total column for eachsample (Figure 2). All region data
can be exported and reported whenselecting the region table. Note that theconcentration shown in the region tableof the TapeStation Analysis software,
as well as in exported files and reports,
refers to the concentration of the defined
region; the total sample concentration isdisplayed in the sample table.

Figure 2. FFPE RNA sample analysis showing DV200 as percentage of total in the region table.

3Results and Discussion

A 70

55
DV200 (%)

55
50
45

3030
Sample 635

Sample 5

35

Average
DV200 (%)

6161

59

Average
DV200 (%)

403651CV (%)

3.0

0.5

0.6

CV (%)

3.9

2.6

1.8

Table 1. Consistency of DV200 analysis with the Agilent RNA and the Agilent High Sensitivity RNA
ScreenTape assay measured in dilution series for three samples per assay.
Agilent RNA ScreenTape assay
Sample 8

Sample 9

Sample 10Sample 11

Average DV200 in %4652425766

64Standard deviation

1.1

1.6

1.7

1.6

0.6

0.9% CV

2.4

3.1

4.1

2.8

1.0

1.4

B
70

65

65

60

60

55

55

50
45

35

35
60
80
100
Concentration (ng/µL)

120

140

160

Agilent High Sensitivity RNA ScreenTape assay

454040Sample 12504020

Agilent High Sensitivity RNA ScreenTape assay

Sample 7

Agilent RNA ScreenTape assay

0454070305040

DV200 (%)

DV200 (%)

60

Figure 3. Reproducibility of DV200 analysis for three individual samples with (A) the Agilent RNA ScreenTape
assay and (B) the Agilent High Sensitivity RNA ScreenTape assay.

The dilution series showed that DV200evaluation is most precise whenmeasuring above the lower limit of thespecified assay concentration range, as
shown in Figure 4.

A60

Sample 4A dilution series for three individual
FFPE RNA samples was analyzed
with the RNA and High Sensitivity
RNA ScreenTape assays to determine
the consistency of the DV200 analysis(Table 1). Both the RNA ScreenTape andHigh Sensitivity RNA ScreenTape assay
yielded comparably high DV200 precisionof less than 5 % CV.

65

Sample 3

Concentration Dependence

65Sample 2

DV200 (%)

A set of three FFPE RNA samples withfive individual replicates was used toverify the reproducibility of the DV200 with
the RNA and the High Sensitivity RNA
ScreenTape assay. Both assays showed
high reproducibility with a CV of less than
5 % (Figure 3).

70

Sample 1

Reproducibility

B

30

0

1,000

2,000

3,000

4,000

5,000

6,000

7,000

8,000

Concentration (pg/µL)

Figure 4. DV200 evaluation of a dilution series of three individual FFPE samples. The red line indicates the lower limit of the assay’s concentration range. Horizontal
lines display the average DV200 of each individual sample. Only data within the respective assay concentration range were used for the evaluation.

4

Severely degraded FFPE samples may
migrate closely to the lower marker
leading to insufficient separation of
the lower marker, especially at sample
concentrations close to the upper limitof the specified quantitative range. In
this case, diluted samples yielded moreaccurate results for DV200 (see Figure 5).
Lower

200

100,000

Lower

200

100,000

3,000A

Sample intensity (norm. FU)

2,500
2,000
1,500
1,000
500

B
2,500
2,000
1,500
1,000
500

4,000
6,000

2,000

1,000

25

4,000
6,000

2,000

1,000

200

500

25

200

00500

Sample intensity (norm. FU)

3,000

Figure 5. Highly degraded FFPE RNA sample merging with the lower marker (A) and with improved marker separation after 1:2 dilution (B).

Assay Comparison

y = 1.0187x
R² = 0.9376
80DV200 (%)
High sensitivity RNA ScreenTape assay

Twenty FFPE RNA samples from various
human tissues were analyzed with theRNA ScreenTape assay, and after 1:20
dilution with the High Sensitivity RNAScreenTape assay. The DV200 obtainedwith the RNA assay ranged from 40 to74 %, and the corresponding samples
analyzed with the High Sensitivity RNA
assay resulted in 101 ± 5 % of thesevalues. To verify the comparability ofDV200 measured with the RNA and HighSensitivity RNA ScreenTape assays, bothvalues were plotted against each other,as shown in Figure 6.

907060504030202030405060708090

DV200 (%) RNA ScreenTape assay

Figure 6. Comparison of DV200 data from FFPE RNA samples obtained with the Agilent RNA ScreenTapeand the Agilent High Sensitivity RNA ScreenTape assays with a linear regression line forced through theorigin.

5Conclusion

Reference

The Agilent RNA and Agilent HighSensitivity RNA ScreenTape assays
together with the Agilent 4200
TapeStation enable fast and easy quality
analysis of FFPE RNA samples. TheAgilent TapeStation Analysis software
displays the DV200 after region setup
as a percentage of total. The RINealgorithm remains unaffected by theDV200 calculation. The DV200 evaluation
with the RNA and High Sensitivity
RNA ScreenTape assays is highly
reproducible, and both assays yield
highly comparable results. For best
performance, it is recommended to run aladder together with the samples. Within
the specified assay ranges, the DV200
is consistent in dilution series. Highly
degraded samples with insufficient
marker separation should be diluted formost accurate results.

1.

www.agilent.com/chem
For Research Use Only. Not for use in diagnostic procedures.
This information is subject to change without notice.
© Agilent Technologies, Inc. 2017
Printed in the USA, September 28, 20175991-8355EN

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.