Agilent 2100 Bioanalyzer System
Agilent 4200 TapeStation System

Applications for DNA, RNA,and Protein Analysis
Application Compendium

2

Agilent 2100 Bioanalyzer System
Agilent 4200 TapeStation System

Applications for DNA, RNA,and Protein Analysis
Application Compendium

3

4

Let us provide you with the right solution
for your success.
Agilent Technologies is a leading global provider of life science andchemical analysis solutions, including instrumentation, supplies,
software, and services. Agilent helps life science investigators advancebasic research and accelerate discovery and development.

Agilent’s life sciences business addresses the needsof academic, institutional, and pharmaceuticalscientists in five key areas:– Genomics– Proteomics– Metabolomics– Bioinformatics– Pharmaceutical research

5

Contents
Agilent 2100 Bioanalyzer system
Introduction Agilent 2100 Bioanalyzer system

10

Microfluidic electrophoresis

11

DNA analysis

14

Next-generation sequencing
DNA library QC in target enrichment andnext-gen sequencing workflows
Sizing and quantitation of DNA libraries andfragmented DNA
Quality control of FFPE DNA samples
Analysis of limited DNA material on the
Pippin Prep system

17

PCR product analysis
Optimize QPCR assay design

18

Food analysis
Estimation of non-basmati rice amounts inbasmati rice products
Strawberry and raspberry fruit differentiation
Detection of non-fish species
with PCR-RFLP

14
15
16

19
20
21

RNA analysis
Analysis of total RNA
Standardization of RNA quality control
Quality control with DV200 evaluation of
FFPE RNA samples
Assessing integrity of plant RNA
Assessing integrity of insect RNA

6

22
23
24
25

Analysis of Small RNA
Analysis of miRNA content in total RNA samples
Analysis of small RNAs from Drosophila Schneider

26
27

Genome editing

28

Guide RNA analysis for CRISPR-Cas9 genome editing

28

Protocol optimization for CRISPR-Cas9 workflows

29

Protein analysis

30

Protein purification
Optimization of on-column cleavage

30

Analysis of modified proteins
Analysis of PEGylated proteins

31

High sensitivity protein analysis
Detection of low abundant proteins
Highly specific and sensitive alternative
to western blotting
Quantitation of low protein abundance withan internal standard

32
33
34

Antibody analysis
Analysis of IgG2 under nonreducing conditions
IgG2 analysis under reducing conditions

35
36

Food analysis
Rapid wheat varietal identification
Protein analysis in milk

37
38

7

Contents
Agilent 4200 TapeStation system
Introduction Agilent 4200 TapeStation system

39

DNA analysis

42

Next-generation sequencing
Impact of gDNA Integrity on the outcome of
DNA methylation studies
Analysis of DNA from FFPE tissue
Quality control of DNA from FFPE tumor samples
Quantitation of amplified SureSelectQXT
whole genome libraries
Quality control of final NGS libraries
Analysis of NGS libraries – equivalence of the4200 TapeStation and 2200 TapeStation system

47

PCR product analysis
Quality control of single cell DNA samples

48

Genomic DNA
Optimization of gDNA Extraction from FFPE tissue
Equivalence of the 4200 TapeStation and2200 TapeStation systems
Quantitation of isolated genomic DNA (gDNA) samples
Quantity and integrity analysis of genomic DNA for
next generation sequencing
Integrity analysis of genomic DNA frombacterial sources

8

42
43
44
45
46

49
50
51
52
53

RNA analysis

54

Gene Expression Analysis
Impact of RNA degradation on qPCR experiments

54

Analysis of total RNA
Equivalence of the 4200 TapeStation and2200 TapeStation system RNA integrity number
Equivalence of the 4200 TapeStation and2200 TapeStation system quantitation
Comparison of the RNA integrity analysis on 2100
Bioanalyzer system with 2200 TapeStation system
Equivalence of RNA analysis on 2200 TapeStationand 2100 Bioanalyzer system

55
56
57
58

RNA Quantitation
RNA quantitation with the TapeStation system incomparison with spectrophotometric and fluorescencebased measurements

59

RNA Purity
Detection of genomic DNA contamination

60

Analysis of FFPE RNA
Simplified DV200 Evaluation with the 4200 TapeStation

61

LIMS Integration

62

LIMS integration of theAgilent 4200 TapeStation system

62

Literature

63

Overview Application Notes and other publications

9

Agilent 2100 Bioanalyzer system
A wide range of applications
The Agilent 2100 Bioanalyzer system is an easy-to-use
benchtop platform with ready-to-run kits for a wide range
of applications.
– DNA size and quantity
High resolution separation and quantitation of
DNA down to pg/μL sensitivity.

Learn more at:www.agilent.com/genomics/
bioanalyzer– RNA quality check with RIN
An established standard for RNA analysis offering
analysis of total RNA, mRNA, and small RNA.
Objective integrity analysis of total RNA is provided
with RIN (RNA Integrity Number) and with the DV200
metric (for FFPE RNA samples prior to RNA sequencing
studies).
– SDS-PAGE replacement for protein analysis
The fast and reliable way to determine the quantityand purity of proteins from Coomassie down tosilver stain sensitivity.
When combined with any Agilent kit, you will discover
how microfluidic technology can revolutionize yourlaboratory. Microfluidics utilizes interconnected networksof microchannels and wells for the analysis of various
sample types. The technology reduces overall space andvolume requirements, and allows online integration of many
workflow steps, such as sample enrichment, separation,
staining, destaining, and detection.
Advantages of microfluidics include dramatically reduced
sample and reagent consumption, significantly faster
analysis times, and reduced hands-on work during sample
preparation and data analysis. The Agilent 2100 Bioanalyzer
system represents the first microfluidic platform forthe electrophoretic analysis of DNA, RNA, and proteins.
This versatility makes the 2100 Bioanalyzer system
an indispensable tool for the molecular biologist andbiochemist.
10Microfluidic electrophoresis increases the quality
and efficiency of your analysis
Microfluidic electrophoresis has many advantages overconventional methods, such as gel electrophoresis.

1.

2.

3.

4.

Principle of operation
1. The sample moves through the microchannels from the sample well.
2. The sample is injected into the separation channel.
3. Sample components are electrophoretically separated.
4. Components are detected by their fluorescence and translated intogel-like images (bands) and electropherograms (peaks).

One of the many benefits the Agilent 2100 Bioanalyzer
system has over conventional bioanalytical methods is theelimination of time consuming procedures—so you canenjoy standardized handling and interpretation of data. It
simplifies the process of data gathering and analysis down
to three quick and easy steps:
Load sample, run analysis, view data
– The Agilent 2100 Bioanalyzer system and the various
DNA kits are the tools of choice for automated sizing
and quantitation of products generated by RT-PCR andany type of multiplex PCR with unprecedented accuracy
and reproducibility. It not only provides the detectionof the presence or absence of a PCR product, but alsooffers quantitation of this product and detection ofunspecific amplification. Therefore, the Agilent 2100
11

Bioanalyzer system helps to optimize PCR reactions
for gene expression, sequencing, cloning, and accurate
analysis of restriction digests.
– The High Sensitivity DNA kit provides sizing andquantitation of DNA fragments and DNA smears in
the 50 to 7000 bp size range down to pg/μL sensitivity.
This is especially useful for sample quality control
and the monitoring of critical steps in next-generation
sequencing (NGS) workflows, including DNA
fragmentation, target enrichment, and DNA library
amplification.
– Due to the omnipresence of RNases and the instabilityof RNA, integrity checks and sample quantitation are
essential steps before any RNA-dependent experiment.
The 2100 Expert software generates the unambiguous
RNA Integrity Number (RIN), provides a quantitation
estimate, calculates ribosomal ratios of total RNA
samples, and automatically detects ribosomal RNA
contamination in mRNA. The RNA 6000 Nano kit
is a well-established standard for RNA sample quality
control. The RNA 6000 Pico kit allows detection of
RNA degradation with sample amounts down to
200 pg of total RNA.
– The high resolution Small RNA kit allows separation,verification, and optimization of miRNA after extraction
procedures. It is also often used for sample quality
control of guide RNAs for CRISPR experiments.
By consuming only 1 μL sample, even pg amounts ofpurified small RNA can be measured reproducibly andcomparably within 30 minutes. By effectively staining
single- and double-stranded oligonucleotides at thesame time, the assay is a versatile tool.

12

– The Protein 80 and Protein 230 kits provide fast andeasy analysis of a wide range of samples, whether
expressed recombinant proteins, purified proteins,stability studies, or an antibody quality check.
Microfluidic electrophoresis provides size, purity,and concentration information for 10 protein samples
in less than 30 minutes. This approach eliminateshandling of SDS-PAGE gels, staining, or imaging steps.
– With the High Sensitivity Protein 250 kit, it is possible
to analyze proteins down to 1 pg/μL (on chip), whichis equivalent or superior to silver stain SDS-PAGE.
It provides quantitation over a dynamic range of up to
four orders of magnitude with the reproducibility andease-of-use only associated with the Agilent 2100
Bioanalyzer system.
– The optional Agilent 2100 Security Pack software
ensures full 21 CFR Part 11 compliance of your 2100
Bioanalyzer system for regulated environments suchas pharmaceutical QA/QC labs or manufacturing. It
addresses requirements, such as electronic signatures,
audit trails, and user authentication. Along with IQ and OQ
support services and declarations of conformity for allcomponents offered for all assays and kits, your Agilent
2100 Bioanalyzer system will be compliant in no time.

13

Next-generation sequencing
DNA library QC in target enrichment and next-gen sequencing workflows

[FU]

A

B
10 cycles

350

[FU]

16 cycles (1:10 dil.)
14 cycles (1:10 dil.)

450
300

400

250

350

12 cycles (1:10 dil.)

300
200
250
150

200

8 cycles

150

100

100
50

6 cycles4 cycles

50

0

0
35

100

200

300

400

600 1000 2000

10380 [bp]

35

100

200

300 400 500 600 1000 2000 10380 [bp]

From
[bp]

To
[bp]

Corrected % of
area
total

Averagesize [bp]Size distribution
in CV [%]

Concentration[pg/µL]

Molarity[pmol/L]

100

2,000

395.8

254

12.4

283.33

1,713.8

51

Quantitation after 4 PCR cycles

Kit:

High Sensitivity DNA kit

Software Assay: High Sensitivity DNA assay
Abstract:
The High Sensitivity DNA kit was used for quality control of amplified and purified
DNA samples from the post-hybridization PCR amplification step before sequencing during the
SureSelect Target Enrichment workflow. The electropherograms of typical PCR amplified DNA libraries
show a smear from 150 to 350 nucleotides. The key observation clearly shown in figure B, is that thequality of the PCR product is dependent on the number of PCR cycles performed. After 14 PCR cycles,
an additional DNA smear at approximately 500 bp was detected in the electropherogram. The highly
sensitive nature of the High Sensitivity DNA kit allowed the amplified DNA to be detected and reliably
quantified, even after only four PCR cycles. Thus, the number of library PCR cycles can be reduced,
removing amplification bias and significantly improving the data quality with increased accuracy.

Application note: 5990-5008EN
14

Next-generation sequencing
Sizing and quantitation of DNA libraries and fragmented DNA

10: library 1:20
8: library 1:10

6: library 1:5
4: library 1:2

2: library 1:20
4: library 1:10

2: library
3: TE blanklibrary 1:20

[FU]
1000

7: library
9: TE blanklibrary 1:20

[FU]
400

A

6: library 1:5
8: library 1:2

B

350
300

DNA library

Fragmented
DNA

250
500

200
150
100
50

0

0
100

200

10

1
Y intercept = 0.09
r = 0.993
0.1

200

300 400 500 600 1000 2000 10380 [bp]10

1
Y intercept = 0.18
r 2 = 0.993
0.1

1
10
Reference concentration [ng/µL]

Kit:

35 100300 400 500 600 1000 2000 10380 [bp]Measured conc. [ng/µL]

Measured conc. [ng/µL]

35

0.1

1
10
Reference concentration [ng/µL]

High Sensitivity DNA kit

Software Assay: High Sensitivity DNA assay
Abstract:
The High Sensitivity DNA kit provides sizing and quantitation of DNA fragments
and DNA smears in the 50 to 7000 bp size range down to pg/μL sensitivity. This is especially usefulfor sample quality control and the monitoring of critical steps in next-generation sequencing (NGS)
workflows, including DNA fragmentation, target enrichment, and DNA library amplification. The analysis
of a dilution series from two typical NGS samples, (A) Illumina DNA library and (B) fragmented DNA was
performed. For both DNA sample types, the double logarithmic plot demonstrates an excellent linearity
with r2 = 0.993. The linear dynamic range for smear samples of the High Sensitivity DNA kit was foundto be between 50–100 pg/μL and 5,000–10,000 pg/μL. This linear dynamic range depends on the library
type and fragment distribution. The broad linear dynamic range of the High Sensitivity DNA kit enables
the detection of less abundant products, such as PCR artifacts and impurities.

Application note: 5990-4417EN
15

Next-generation sequencing
Quality control of FFPE DNA samples

1. FFPE DNA Q[FU]

2. FFPE DNA A

3. Frozen tissue DNA

4. Control DNA

300
250
200
150
100
50
0
15 50 100

Sample200300

400 500

700

1500

Average size [bp]Peak height [bp]FFPE DNA Q

307

264

Concentration [ng/µL]
34.8

FFPE DNA A

309

254

22.6

Frozen tissue DNA

331

264

89.5

Control DNA

348

271

23.2

Average size, peak height, and quantification of precaptured amplified libraries.

Kit:

DNA 1000 kit, High Sensitivity DNA kit

Software Assay: DNA 1000 assay, High Sensitivity DNA assay
Abstract:
The Agilent 2100 Bioanalyzer system was used for quality control of DNA samples
from formalin-fixed paraffin-embedded (FFPE) and fresh-frozen tissues before and during the
SureSelect Target Enrichment workflow. The figure shows the electropherogram overlay of precapture
amplified samples after five PCR cycles run on the Agilent 2100 Bioanalyzer system with the Agilent DNA
1000 kit. Similar profiles were observed for all DNA samples. No amplification artifacts or primer dimers
were seen. FFPE DNA samples gave comparable results to DNA from fresh-frozen tissue and control
cell line DNA, appropriate for downstream sequencing on the Illumina platform.
Reliable DNA electrophoresis with the Agilent 2100 Bioanalyzer system provided smear profiles anddetails for library statistics, such as peak heights, average smear size, size distribution, and DNA
concentration.

Application note: 5991-0483EN
16

Next-generation sequencing
Analysis of limited DNA material on the Pippin Prep system

[FU]
160
140
120
100

DNA 100080

100 ng

60
40
20

50 ng

0
15

50

100

150

200

300

400

500

700 1000 1500

bp

10380

bp

[FU]

120

High Sensitivity DNA

100
80
60

1.2 ng0.625 ng40

0.375 ng

20
0

35

Kit:

100 150 200300

400 500 600

1000 2000

DNA 1000 kit, High Sensitivity DNA kit

Software Assay: DNA 1000 assay, High Sensitivity DNA assay
Abstract:
The Agilent 2100 Bioanalyzer system and the High Sensitivity DNA kit complement
the Pippin Prep automated size selection workflow (Sage Science, Inc.). The figure demonstrates the
DNA analysis of a restriction digest of E. coli genomic DNA to simulate a sheared Pippin Prep input
sample. Using the High Sensitivity DNA kit allows the use of only 1.2 ng DNA to achieve a roughly
equivalent signal to 50 ng on the DNA 1000 kit. Even lower DNA amounts (0.375 ng) yield reasonable
electropherograms showing input size distribution. This allows tailoring fractionation settings for
the Pippin Prep system, and maximizing chances for successful library construction. Afterwards,
the 2100 Bioanalyzer system can be used to confirm size ranges, quality, and purity of the Pippin
Prep process. The 2100 Bioanalyzer and Pippin Prep systems work well in concert to enable finecontrol and increase the efficiency of library generation before next-generation sequencing.

Application note: 5990-8382EN
17

PCR product analysis
Optimize QPCR assay designAoligo-dT

Size: 110 bp

randomB

Size: 121 bp

oligo-dT
randomB

A

Size: 21 + 51 bp

NTC

Kit:

DNA 1000 kit

Software Assay: DNA 1000 assay
Abstract:
To determine the best possible reverse transcription (RT) priming strategy, QPCR,
and 2100 Bioanalyzer system tests were conducted for three genes (GAPDH, HPRT1, and YWHAZ) byusing high-quality RNA (RIN = 10) as template. The RNA was reverse transcribed with either oligo-dT or
random priming (A: 5’-assay, B: 3’-assay for GAPDH). To assess size and purity of QPCR amplicons, 1 μLof the QPCR reactions was analyzed with the DNA 1000 assay. In addition, no-template-controls (NTC)
were used to assess contamination and potential primer dimer formation, since a small amount ofcontaminating template can lead to amplification. For the HPRT1 5’ oligo-dT assay one of the NTC was
positive, showing a peak in the melt curve at a similar melting temperature compared to the positive
control (A). To verify that no contamination of the well has occurred, the NTC was analyzed using the
DNA 1000 assay on the 2100 Bioanalyzer instrument (B). Two minor peaks could be detected at 21 and51 bp, which are most probably related to primer and primer dimers. This highlights the high information
content obtained by the 2100 Bioanalyzer system and the poor discrimination capabilities of a SYBRGreen-based melt curve.

Application note: 5989-7730EN
18

Food analysis
Estimation of non-basmati rice amounts in basmati rice products

RM339 PCR with samples containing 0% to 40% (w/w) Pak 386(non-basmati) in basmati

10%

0%

20%

40%

Unknown sample

Kit:

DNA 1000 kit

Software Assay: DNA 1000 assay
Abstract:
Ensuring integrity of raw food materials, ingredients, and products is both a productquality and regulatory compliance concern. Food ingredient suppliers, manufacturers, and retailers
can suffer economic and legal damages if proven to be supplying materials or products that areincorrectly labeled due to substitution or contamination. For example, EU Commission 1549/04
grants lower import tax on nine basmati rice varieties. Furthermore, the level of non-basmati rice ina basmati rice product must not exceed 7 %. Therefore it is required to identify basmati rice varietiesand determine the level of non-basmati rice in basmati rice products. The 2100 Bioanalyzer systemand the DNA 1000 kit can be used as a quick and cost effective analytical method to differentiate
approved and nonapproved rice varieties using three primer sets and to estimate the level of nonbasmati rice using reference rice admixtures. Four reference samples with 0 to 40 % non-basmati rice
content were used to estimate the non-basmati amount in an unknown sample to be 10-20 %.

Application note: 5989-6836EN
19

Food analysis
Strawberry and raspberry fruit differentiation

Strawberry sauce [30 ng DNA]

Strawberry sauce [30 ng DNA]

Overall results for sample 1:
Number of peaks found:Peak table for sample 1:

Overall results for sample 1:
Number of peaks found:Peak table for sample 1:

Peak
1
2
3
4
5
6

Kit:

Size
[bp]
15
122
134
138
282
1,500

Strawberry sauce
4
Strawberry sauce

Conc.
Molarity[ng/µL] [nmol/L]
4.20
424.2
4.51
56.1
0.59
6.7
0.64
7.0
1.69
9.1
2.10
2.1

Observations
Lower marker

Peak
1
2
3
4

Size
[bp]
15
143
161
1,500

Strawberry sauce
2
Strawberry sauce

Conc.
Molarity[ng/µL] [nmol/L]
4.20
424.2
3.71
39.4
3.01
28.2
2.10
2.1

Observations
Lower marker

Upper marker

Upper marker

DNA1000 kit

Software Assay: DNA 1000 assay
Abstract:
Food authentication is important to help detect fraudulent replacement of expensive
food ingredients, and to ensure correct ingredient content in prepacked foods. A PCR method followedby automated electrophoresis with the Agilent 2100 Bioanalyzer system and the DNA 1000 kit was used
to distinguish strawberry and raspberry samples.
The figure shows the characteristic profiles obtained by the analysis of strawberry sauce through PCR
with the microsatellite primer pairs Fvi11 and Fvi20. This method allows clear differentiation between
samples that contain strawberry (Fragaria) and raspberry (Rubus) DNA and may prove useful in foodauthentication.

Application note: 5990-3327EN
20

Food analysis
Detection of non-fish species with PCR-RFLP
Pork/wild bear

Sample 1

Beef

Sample 2

Lamb

Sample 1

Kit:

Sample 1

Sample 2

TurkeySample 2

Sample 1

Sample 2

DNA 1000 kit

Software Assay: DNA 1000 assay
Abstract:
A PCR-Restriction Fragment Length Polymorphism (RFLP) method was developed
to identify fish species from processed foods using a cytochrome B PCR target sequence withanalysis of the restriction pattern fragments on the Agilent 2100 Bioanalyzer system with DNA 1000
assay. Feasilbility for detection of non-fish species, especially mammalian or avian DNA originatingfrom dairy products or meat, was tested. The assay was successfully used to differentiate pork, beef,
and lamb. Pork and wild boar resulted in the same pattern. The figure shows the restriction digestion
analysis on the Agilent 2100 Bioanalyzer system and the DNA 1000 assay. The gel-like images
show the typical pattern obtained after digestion with three restriction enzymes (Dde I, Hae III,and Nla III) for two meat samples per species. Of avian samples, only turkey DNA resulted in easily
identifiable patterns.

Application note: 5990-8452EN
21

Analysis of total RNA
Standardization of RNA quality control
RIN

8.7 8.8 8.4 7.5 6.4 5.0 4.5 3.7 3.6 2.6 2.5 2.3

Incubation time (increasing degradation of RNA)Intact RNA:RIN 10

28S
28S

18S

18S

FU

Partially degraded RNA:
RIN 5

Strongly degraded RNA:
RIN 3

19

Kit:

24

29

34

39

44
49
Time [s]

54

59

64

69

RNA 6000 Nano kit

Software Assay: Eukaryote Total RNA Nano assay
Abstract:
The RNA integrity number (RIN) is calculated by a dedicated software algorithm toassess the quality of RNA preparations. The RIN tool is a major step in the standardization of userindependent RNA evaluation and delivers more meaningful information than simple ratio calculations
for ribosomal RNA peaks. It is not influenced by instrument, sample integration, and most importantly,
concentration variability, thereby facilitating the comparison of samples and avoiding cost-intensive
experiments with low quality RNA preparations. The RIN algorithm is based on a large collection of RNA
data of various tissues and qualities. Furthermore, anomalies like genomic DNA contaminations areindicated with weighted error messages (critical/noncritical) to achieve maximum reliability.

Application note: 5989-1165EN
22

Analysis of total RNA
Quality control with DV200 evaluation of FFPE RNA samples

Kit:

RNA 6000 Nano and Pico kits

Software Assay: DV200 RNA Nano and Pico assays
Abstract:
With the DV200 metric, degraded RNA samples extracted from FFPE tissue canbe classified according to their size distribution. This Technical Overview describes the simplified
evaluation of the DV200 using the DV200 RNA Nano and DV200 RNA Pico assays with the Agilent 2100
Bioanalyzer system. The assays can be used for re-analysis of existing data files as well as for new
data acquisition. The DV200 RNA assays automatically define a region from 200 to 8000 nucleotides
as shown in the figure. A corresponding DV200 is provided in the Region Table in the column % of Total.
The DV200 results can be saved, exported, and displayed in reports. Samples with RIN <4 are flagged
in different colors for easy assessment of the DV200 range.

Technical Overview: 5991-8287EN
23

Analysis of total RNA
Assessing integrity of plant RNA

40

30
25S

20
15

18S

25S

35

Sunflower(root)
RIN = 8.4

18S

30
Fluorescence

Fluorescence

25

Marker10
525

Sunflower(leaf)
RIN = 6.6

20
15 Marker
10
5

0
_

0
_

5

80

90
25S

80
60
50

60
Fluorescence

Fluorescence70

Lettuce
RIN = 7.8

70

Kit:

5
18 23 28 33 38 43 48 53 58 63 68
Time [s]18 23 28 33 38 43 48 53 58 63 68
Time [s]

18S

40
30

25S
18S

PotatoRIN = 7.6

50
40
30
20

20 Marker
1010 Marker

0_10
18 23 28 33 38 43 48 53 58 63 68
Time [s]_10
18 23 28 33 38 43 48 53 58 63 68
Time [s]0RNA 6000 Nano kit

Software Assay: Plant RNA 6000 Nano assay
Abstract:
High-quality RNA, free of genomic DNA, is a critical determinant for the successof many downstream techniques used in functional genomics, such as RT-PCR and microarraybased experiments. The dedicated Agilent 2100 Bioanalyzer plant RNA assay included in the 2100
Expert software (version B.02.07 or higher) allows rapid assessment of plant RNA integrity frommultiple plant sources and differing degradation states with excellent precision. The figure showsthe electrophoretic separation of different plant total RNA using the RNA 6000 Nano kit with theplant RNA assay. In all samples, the abundant 25S and 18S ribosomal RNA peaks are well resolved
and automatically identified by the software. Compared to the root samples, the leaf and lettuce
extracts exhibit additional fast migrating peaks corresponding to smaller chloroplast ribosomal
RNAs showing that total RNA profiles can vary depending on species and tissue types.
The plant RNA assay and the RIN algorithm provide a convenient, user-independent assessmentof total plant RNA integrity.

Application note: 5990-8850EN
24

Analysis of total RNA
Assessing integrity of insect RNA

Kit:

RNA 6000 Nano and Pico kits

Software Assay: Eukaryote total RNA Nano assay
Abstract:
This Application Note describes the complexity of insect RNA and the challenges
associated with integrity determination using the 2100 Bioanalyzer system. Here, various extracts ofinsect species were analyzed for total RNA quality using the 2100 Bioanalyzer system and the obtained
RNA profiles and RIN were compared. Figure A shows representative RNA profiles of D. melanogaster(C), A. mellifera (E), C. floridanus (F), Z. nevadensis (G), H. zea (H), and M. persicae (L). The effect of degradedinsect RNA on the outcome of quantitative RT-PCR experiments was evaluated with Figure B showing
degradation of total RNA samples from D. melanogaster at room temperature for the indicated time.
Even though the RIN, developed for eukaryotic total RNA, may not be practical for all insect species,
the electropherograms provide suitable information to judge RNA integrity across insect species.

Application note: 5991-7903EN
25

Analysis of small RNAs
Analysis of miRNA content in total RNA samples

A

C

RIN
Total RNA
concentration% of miRNA in total
RNA samples

B

tRNA

Small rRNA

miRNA
concentration
miRNA/SmallRNA
ratio

miRNA

Kit:

Small RNA and RNA 6000 Nano kit

Software Assay: Small RNA and Eukaryote Total RNA Nano assay
Abstract:
Several total RNA samples containing small RNAs (including miRNAs, siRNA, andsnRNA) were analyzed using the RNA 6000 Nano assay to determine concentration and quality,
including RIN. All total RNA samples were then analyzed with the Small RNA assay to measure miRNAconcentration. The relative amount of miRNA was manually calculated as a ratio of the concentrationof miRNA in total RNA.
A) total RNA analyzed with the RNA 6000 Nano assayB) small RNA analyzed with the Small RNA assay
C) miRNA analysis workflow

Application note: 5989-7870EN
26

Analysis of small RNAs
Analysis of small RNAs from Drosophila Schneidertransfer RNA

10 ng
flashPAGE
smallRNA2S RNAs
small RNAs
incl. miRNA

5S RNA
5.8S RNA

Kit:

Small RNA kit

Software Assay: Small RNA assay
Abstract:
Small RNA samples were prepared from Drosophila Schneider cells according todifferent protocols and were analyzed using polyacrylamide gel electrophoresis, northern blot, or the2100 Bioanalyzer system. Only 10 ng of total RNA was run on the Small RNA kit. Two sharp peaks,
corresponding to 5.8S and 5S RNA, were clearly resolved. Transfer RNA (tRNA) formed a third large
peak. A small well-defined peak at 30 nucleotides indicated that 2S RNA could be detected with only
10 ng of total RNA. Therefore, the Small RNA kit was significantly more sensitive than a denaturing
15 % polyacrylamide gel stained with ethidium bromide or SYBR Green II, respectively. Hence, theSmall RNA kit appeared to be the method of choice to assess small RNA species contained in a total
RNA preparation.

Application note: 5989-8539EN
27

Genome Editing
Guide RNA analysis for CRISPR-Cas9 Genome EditingA[nt]

Ladder

E1-ZFP42

E2-ZFP42

E3-ZFP42 VE1-ZFP42 VE2-ZFP42 VE3-ZFP42 E1-GUSB

E2-GUSB

E3-GUSB

VE1-GUSB VE2-GUSB VE3-GUSB [nt]

150

150

100

100

80

80

60

60

40

40

20

20

4

4

Gel image showing extended design gRNA sample migrating around 90 nt.

B
[FU]

50

80

Upper marker
10,380bp

LM

70
60

Cleavage
product 1
2,720 bp
Uncleaved
amplicon3,618 bp50
40
30

Cleavage
product 2
978 bp

20
10
0
50

100

300

500

700

1,000

2,000

5,000

10,380 [bp]Electropherogram showing the Cas9 nuclease assay using M2-GUSB gRNA.

Kit:

Small RNA and DNA 7500 kits

Software Assay: Small RNA and DNA 7500 assays
Abstract:
This study demonstrates the seamless integration of Agilent solutions for guide RNA(gRNA) design, in vitro gRNA synthesis, and confirmation of gRNA-directed cleavage efficiency. Several
gRNA sequences were designed using the Agilent SureDesign web-based application. The in vitro gRNA
synthesis was carried out using the SureGuide gRNA synthesis kit. The transcribed and purified gRNAs
were analyzed on the 2100 Bioanalyzer system with the Small RNA kit to assess the size, purity, andquantitation of each gRNA (Figure A) with a high level of accuracy and precision. The efficacy of thesynthesized gRNAs was evaluated using the Agilent SureGuide Cas9 Nuclease kit. Successful gRNAtargeted cleavage of designed PCR amplicons by the Cas9 nuclease yields two fragments of expected
sizes. The fragments were analyzed using the DNA 7500 kit to check for gRNA efficacy as presented
in Figure B. The 2100 Expert software provides the areas of each peak for easy calculation of cleavage
efficiency.

Application note: 5991-7557EN
28

Genome Editing
Protocol optimization for CRISPR-Cas9 workflows
A
Synthesis of gRNA

gRNA Design

Cas9 Nuclease assay

Agilent SureDesign
SureGuide Wizard

Extended design oligos

Primer design

gRNA target sequencesFill-in reaction andpurification

PCR amplification
andpurification

dsDNA template

DNA template

Addition of guidesequences

Minimal design oligos

DNA 7500 Kit

DNA 1000 Kit
Minimal design oligos

Extended design oligos

Cas9 Nuclease digestion
In vitro gRNA transcription

Purified gRNA
Digested fragments

DNase Digestion

DNA 7500 Kit

Small RNA Kit
gRNA purification

Purified gRNA
Small RNA Kit

Schematic diagram showing gRNA design, synthesis, and Cas9 assay workflow. The use of the 2100 Bioanalyzer sytem
for quality control is shown as green, for efficacy assessment as blue and the grey fields indicate where it is suitable for
optimizing the protocol.

Kits:

DNA 1000, Small RNA, and DNA 7500 kits

Software Assay: DNA 1000, Small RNA, and DNA 7500 assays
Abstract:
This study demonstrates the seamless integration of Agilent solutions for guide
RNA (gRNA) design, in vitro gRNA synthesis, and confirmation of gRNA-directed cleavage efficiency
with the overall workflow and quality checks highlighted in Figure A. In addition to quality control andefficacy assessment, the Agilent 2100 Bioanalyzer system can also be used to optimize the protocol
at intermediate steps as indicated in Figure A. A few of these steps include:– Extended design fill-in reaction – Verify the presence of successful fill-in products using the DNA
1000 kit and further optimize the reaction before in vitro transcription.
– Guide RNA purification – Check for successful in vitro transcription by running samples on the
Small RNA kit before the final purification step. Optimize the input oligo concentration, yield, andincubation times.
– PCR amplification of the DNA template – gRNA specificity for the Cas9 nuclease assay requires
PCR amplification of a designed amplicon. Confirm the correct size and yield with the DNA 7500
kit after PCR amplification, before use in the Cas9 nuclease assay.

Application note: 5991-7557EN
29

Protein purification
Optimization of on-column cleavage

Unoptimized on-column cleavage
Optimized on-column cleavage

CleavedproteinTaggedprotein

Kit:

SampleSize [kDa]Rel. Conc. [ng/µL]% TotalUnoptimized on-column
cleavage

71.9
81.9

310.2
414.7

38.3
51.3

Optimized on-column
cleavage

70.9
83.2

993.0
11.4

78.0
0.9

Protein 230 kit

Software Assay: Protein 230 assay
Abstract:
Monitoring the protein of interest (POI) during purification in a fast and reliable
manner is fundamental. Here, protein purification of a His6-Sumo3-tagged DNA-binding protein was
performed with Immobilized Metal Affinity Chromatography (IMAC). The POI was N-terminally fused
to His6-Sumo3-tag, bound to Ni beads, and released from the tag and beads by treatment with Histagged SenP2 protease. Although the protease was used in excess for on-column cleavage, most of
the protein was still found to be fused to its tag. Therefore, the on-column cleavage step was optimized
by determining the ideal ratio of protein-to-beads-to-protease by monitoring the percentage of cleaved
protein versus its tagged precursor with the Protein 230 assay. The molecular weight difference ofabout 10 kDa is sufficient to achieve a baseline separation between the tagged and tagless protein. As
illustrated in the blue electropherogram, almost all tagged protein can be cleaved under the optimized
conditions.

Application note: 5990-6153EN
30

Analysis of modified proteins
Analysis of PEGylated proteins
mPEG - pNP

mPEG - Tresylate
Control

6 mg /mL

4 mg /mL

2 mg /mL

1 mg /mL

LadderLadder

0.5 mg /mL

1 mg /mL

2 mg /mL

4 mg /mL

6 mg /mL

ControlSize [kDa]

97
66

A

44
29

20
14

240.0

150.0

95.0

B

63.0
46.0
28.0
15.0
7.0
4.5

Lane

Kit:

1

2

3

4

5

6

7

8

9

10

11

12

13

Protein 230 kit

Software Assay: Protein 230 assay
Abstract:
Monitoring the degree of PEGylation is important to achieve a balance between retentionof bio-activity, stability, and immunogenicity of the PEGylated species. SDS-PAGE is routinely used
for PEGylation analysis, however, it cannot precisely identify the extent of the PEGylation. In addition,
it can be tedious and time-consuming. The Agilent 2100 Bioanalyzer system was evaluated as analternative to SDS-PAGE. A chimeric protein was subjected to PEGylation at 30 °C for 3 hours using
different concentrations of two PEGylating reagents, mPEG-pNP (mol wt 5000) and mPEG-tresylate(mol wt 5000). Levels of PEGylation were monitored by SDS-PAGE (A) and the 2100 Bioanalyzer
system (B). The profiles of PEGylated protein obtained with the Agilent 2100 Bioanalyzer system weresuperior and allowed differentiation between levels of PEGylation. The Agilent 2100 Bioanalyzer withProtein 230 assay is an easy-to-use tool that provides high resolution of PEGylated protein species,
allowing efficient optimization of reaction conditions as well as fast and quantitative monitoring ofproduction batches.

Application note: 5990-9593EN
31

High sensitivity protein detection
Detection of low abundant proteinsLowermarkerLower
marker

4.2 pg/µL
zoom

4.2 ng/µL

Lower
markerLower
marker0,42 pg/µL
zoom
42 pg/µL
zoom

10000

Relative concentration [pg/µL]

R² = 0.995
1000
R² = 0.999
100
10
1
Individual labelingControl series
0.1

0.1

Kit:

1

10
100
Target concentration [pg/µL]

1000

10000

High Sensitivity Protein 250 kit

Software Assay: High Sensitivity Protein 250 assay
Abstract:
For highest sensitivity, SDS-PAGE gels are commonly silver stained using alaborious procedure with low reproducibility and insufficient quantitation capabilities. The HighSensitivity Protein 250 kit is a superior alternative to silver staining protocols due to reproducible
and fast staining, automated separation and data analysis. It overcomes the critical limitationsof traditional silver staining of SDS-PAGE gels by providing high sensitivity and a linear dynamic
quantitation range of four orders of magnitude combined with excellent reproducibility. The kit
analyzes proteins from 10 to 250 kDa down to an on chip concentration of 1 pg/μL. It is based on
the detection of fluorescently-labeled proteins that are separated by automated electrophoresis.

Application note: 5989-8940EN
32

High sensitivity protein detection
Highly specific and sensitive alternative to western blotting

1

2

3

4

5

6

7
kDa
260
160
110
80
60
50
40
30
20

5: 0.0001 % PTEN
1: 1 % PTEN
6: E. coli only
2: 0.1 % PTEN
3: 0.01 % PTEN 7: PTEN only
4: 0.001 % PTEN

Kit:

High Sensitivity Protein 250 kit

Software Assay: High Sensitivity Protein 250 assay
Abstract:
A targeted protein analysis employing immunoprecipitation in combination with theHigh Sensitivity Protein 250 assay (IP/HSP250 method) was evaluated. Sensitivity and specificity of
the IP/HSP250 method were investigated using E. coli lysate spiked with GST tagged phosphataseand tensin homolog (PTEN). Samples were first fluorescently labeled, then immunoprecipitated,
and finally the complexes formed were directly eluted from magnetic Protein A beads and analyzed
with the High Sensitivity Protein 250 assay. Electropherograms of samples with 0.1 % to 0.0001 %PTEN and a negative control are shown. The zoom shows the main peak of 0.001 % PTEN. The limit
of detection was determined to be 0.001 % or 100 pg PTEN in 10 μg E. coli lysate. For comparison,western blots were performed. The PTEN blots showed a high nonspecific background in all lanes,
due to the secondary antibody. A specific band was observed only at 1 % or 100 ng PTEN in
10 μg E. coli lysate.
Thus, the IP/HSP250 method showed both higher sensitivity and specificity than the western blot,
resulting in a 1,000 fold lower limit of detection for PTEN.

Application note: 5990-4097EN
33

High sensitivity protein detection
Quantitation of low protein abundance with an internal standard
0.9 mg/ml BSA, 20 ng/u...

0.9 mg/ml BSA, 20 ng/u...

160
140

4000

120

BSA
monomer

3500

100
80
60

3000

40
20

2500

0
15

[kDa]

2000
1500
1000

Lower marker

BSA multimers
βLG

500
0

5

15 28

Peak
bLG
BSA monomer

Kit:

AVERAGE% CV
AVERAGE
% CV

95

46 63

150

240

[ kDa]

Size [ kDa]

Rel. Conc. [ pg/ µL]% Total

16.9
1.4
68.7
0.8

184
2.3
4191
3.1

3.3
1.5
75.7
0.3

High Sensitivity Protein 250 kit

Software Assay: High Sensitivity Protein 250 assay
Abstract:
The High Sensitivity Protein 250 assay is based on the detection of fluorescentlylabeled proteins that are separated by automated electrophoresis. The quantitation and sizing of sample
peaks is done relative to a ladder as an external standard on the same chip, in contrast to the Agilent
Protein 80 and Protein 230 assays where the quantitation is based on internal standards. With theHigh Sensitivity Protein 250 kit, it is also possible to do quantitation based on the addition of a suitableinternal standard. In this example, a small standard protein, ß-Lactoglobulin (ßLG, 18.4 kDa), wasused. ßLG was spiked into a BSA solution before the labeling reaction and the analysis was performed
according to the standard protocol. The electropherogram shows an overlay of 10 runs and a zoom onthe ßLG peak in the insert. The quantitation data for the ßLG and BSA monomer is summarized in thetable. Quantitation reproducibility is improved using an internal standard, because well-to-well variations
for sample injection and matrix influences during staining are excluded.

Application note: 5989-8941EN
34

Antibody analysis
Analysis of IgG2 under nonreducing conditions
A

P230

Lower
marker[FU]
250

Systempeak

Intact antibody

200
150
100
Mixture of
LC and HC

50

Upper
marker
ngAB

0
-50
15

20

25

30

35

40

45

50

Time [s]B[FU]

HSP-250

Intact antibody

350

250

Lower
marker150
Mixture of
LC and HC

50

ngAb
Aggregates

-50
15

20

25

30

35

40

45

50

Time [s]Kit:

Protein 230 and High Sensitivity Protein 250 kits

Software Assay: Protein 230 and High Sensitivity Protein 250 assays
Abstract:
Human myeloma IgG2 was analyzed with the 2100 Bioanalyzer system and theProtein 230 (P230) and High Sensitivity Protein 250 (HSP-250) assay under nonreducing conditions.
One representative electropherogram per assay is shown. The intact IgG2 antibody is detected at
156.6 kDa, which is in close agreement with its theoretical molecular mass of about 150 kDa. A
unique feature of the HSP-250 assay is the size and concentration measurement beyond the size
range of the ladder, that is, 250 kDa. Therefore, high molecular weight aggregates or impurities above
250 kDa are sized and quantified as well. The P230 and HSP-250 assays clearly resolve light chain(LC), heavy chain (HC) and a mixture of LC and HC peaks including the nonglycosylated form of IgG2
(ngAb).

Application note: 5990-5283EN
35

Antibody analysis
IgG2 analysis under reducing conditions

A

B
[FU]

P80

[FU]

System
peak

700

HC

500

600

400

500

LC

HC

300

400
300 Lower
marker
200

200

Upper
markerLC

UppermarkerLow MW
impurities

100

P230

Lower
marker
Systempeak

100
Aggregates
0

0
-100

-100
15

15

20

25

30
35
Time [s]

40

45

50

15

20

25

30
35
Time [s]

40

45

50

C
[FU]

HSP-250

HC

350
300
Lower
marker

250
200

LC

150
100
Aggregates

50
0
-50
15

Kit:

20

25

30
35
Time [s]

40

45

50

Protein 80, 230 and High Sensitivity Protein 250 kits

Software Assay: Protein 80, 230 and High Sensitivity Protein 250 assays
Abstract:
Human myeloma IgG2 was analyzed with the 2100 Bioanalyzer system and theProtein 80 (P80), the Protein 230 (P230), and the High Sensitivity Protein 250 (HSP-250) assay in thepresence of dithiothreitol (DTT) as reducing agent. One representative electropherogram per assay isshown. Under reducing conditions, the IgG2 light chain (LC) and heavy chain (HC) are well resolved
with all three available protein assays. Aggregates of higher molecular weight are observed with theP230 and HSP-250 assays whereas the P80 assay resolves low molecular weight (mol wt) impurities
associated with the IgG2 sample.

Application note: 5990-5283EN
36

Food analysis
Rapid wheat varietal identificationα-, β-, γGliadinsA

HMW-Gluteninsω -Gliadins,
L MW-Glutenins

Lower
MarkerUpper
MarkerB

Glasgow_3
Glasgow_2
Glasgow
Glasgow_1
Brompton_2
Brompton_1
Brompton
Ambrosia_2
Ambrosia_1
Ambrosia

Similarity Scale

Kit:

Protein 230 kit

Software Assay: Protein 230 assay
Abstract:
Total wheat proteins (including high molecular weight glutenins, HMW) wereextracted from individual grains. The extract samples were separated on the 2100 Bioanalyzer system.
The Protein 230 assay produced well-resolved protein profiles, suitable for varietal discrimination(A). Electropherogram profiles were processed using the Phoretix 1D Advanced and 1D Database(Nonlinear Dynamics) software for pattern-matching purposes. Replicates of three different wheat
varieties can be correctly grouped in a dendrogram (B). The study has demonstrated that using
the 2100 Bioanalyzer system with the Phoretix system offers a standardized, objective method for
rapid varietal discrimination. The ease-of-use and short analysis times of less than one hour fromsampling to 2100 Bioanalyzer system result makes it most suitable for mill intake use.

Application note: 5989-7735EN
37

Food analysis
Protein analysis in milkA

Cows milk

[FU]
2000

1500
system
peak
1000

500

lower
markerupper
marker0
1.6 3.5

6.5

15

28

46

63

B

95

[kDa]

Cows milkb-casein[FU]

7: cow milk
2: a-casein
3: b-casein
4: k-casein

2000
a-casein

5: a-lacalbumin
6: b-lactoglobulin

1500

1000

500

system a-lactalbuminpeakb-lactoglobulin
lower
marke
r

k-caseinuppermarker0
1.6 3.5

Kit:

6.5

15

28

46

63

95

[kDa]

Protein 80 kit

Software Assay: Protein 80 assay
Abstract:
Qualitative and quantitative information about the main protein species in milk is an
important quality criterion. Cow, goat, and sheep milk were analyzed by automated electrophoresis
with the Agilent Protein 80 kit and the Agilent 2100 Bioanalyzer system. The figure shows the analysis
of commercially available cows milk. The five major milk proteins were analyzed separately andoverlaid with the milk electropherogram to identify the milk proteins. The electropherograms allow
an easy and reliable comparison of different samples. Furthermore, milk from different species could
be distinguished based on their protein pattern which facilitates incoming inspection in routine labs.
The Agilent 2100 Bioanalyzer system and the Protein 80 kit provide a fast, reproducible, and robust
qualitative and quantitative method for milk analysis.

Application note: 5990-8125EN
38

Agilent 4200 TapeStation system
Building on the success of the 2100 Bioanalyzer system,
the Agilent 4200 TapeStation system offers 96-well platecompatibility combined with scalable throughput. Thesefeatures make it an ideal solution for quality controlof biological samples in next-generation sequencing,
microarray, and qPCR workflows in high throughout
applications. The system offers walk away operation withfully automated sample processing. Any sample number
from 1 up to 96 samples can be analyzed at constantcost-per-sample.
Learn more at:www.agilent.com/genomics/
tapestation– DNA size and quantity
Quality control of NGS libraries and genomic DNA
samples– RNA quality check with RINe
Integrity and quantitation of euk. and prok. total RNA
samples
The 4200 TapeStation system uses credit card-sized,
ScreenTape consumables that are available for DNA, andRNA and applications. Sample analysis has never been soeasy – simply load the 4200 TapeStation instrument with
ScreenTape consumable, loading tips, and your samples
in 2 x 8 tube strips or a 96 well plate. An integrated pipettetransfers the samples onto the ScreenTape device. After
automated electrophoresis and imaging, results areavailable in as little as 1 minute per sample. The ready-touse ScreenTape technology enables ultimate flexibility forswitching between assays as well as sample preparation.
With sensitive detection and zero carryover, you get
the utmost confidence in your results, ensuring yourdownstream workflow is a complete success.
The 4200 TapeStation software includes functionality
for instrument control and data analysis. It is intuitive andpresents the automatically analyzed data as familiar gel
image and electropherogram for each sample. With a few
mouse-clicks the software generates customized reports
or enables data export in various different formats.

39

Additional benefits– Unused lanes on the ScreenTape consumable can beused at a later point of time.
– Achieve user-independent results with minimal manualintervention and excellent reproducibility for sizing,
concentration, and integrity assessment.
– Requires only 1-2 μL of DNA or RNA sample per run, evenfor high sensitivity analysis.
– Rely on the integrity standards for total RNA (RNAIntegrity Number equivalent, RINe) and genomic DNA(DNA Integrity Number, DIN).
– Carryover is eliminated as the ScreenTape consumableanalyses each sample in a separate lane with individualloading tips.
– Sample evaporation is avoided by covering the 96-wellplate with pierceable foil.
– Save valuable bench space in your lab with a compact
system offering the complete sample QC solution.
The D1000 ScreenTape assay facilitates the separation
and analysis of DNA fragments, fragmented DNA, and NGS
libraries from 35 - 1,000 bp. Choose between the D1000 andthe High Sensitivity D1000 ScreenTape assay dependingon the sensitivity requirements of your application. Bothassays allow accurate and reliable sizing and quantitation.
The High Sensitivity ScreenTape assay requires only 2 μL
sample input and has a sensitivity of 5 pg/μL per fragment.
ScreenTape consumables, sample buffer, as well as ladder
are available as standalone products.
The D5000 ScreenTape assay allows for the separation
and analysis of DNA fragments from 100 - 5,000 bp, ideal
for larger sized NGS libraries, PCR products, or restriction
digests. Depending on the expected sample concentration,
it is possible to choose between the D5000 ScreenTape andthe High Sensitivity D5000 ScreenTape application.
40

The Genomic DNA ScreenTape assay was developed
for the separation and analysis of genomic DNA (gDNA)
from 200 to greater than 60,000 bp. It provides accuratequantitation and sizing data as well as an automatednumerical assessment of gDNA quality based on the
DNA integrity number (DIN) and is therefore the ideal
QC tool for next generation sequencing (NGS) and arraycomparative genomic hybridization (aCGH) workflows.
The DIN algorithm is included in the 4200 TapeStation
software, and provides a numerical assessment of the
DNA sample by assigning a numerical score from 1 to
10. A high DIN indicates highly intact gDNA, and a low
DIN a strongly degraded gDNA sample. The Genomic
DNA ScreenTape assay allows sample quantitation in therange of 10-100 ng/μL. The functional range for the DNA
integrity number is specified between 5 and 300 ng/μL.
The RNA ScreenTape assay provides efficient andreliable RNA analysis for RNA characterization andquality assessment. The RNA integrity number equivalent(RINe) delivers an instant and objective evaluation of RNA
degradation for eukaryotic and prokaryotic total RNA
samples. The RINe number is directly comparable to thewidely accepted and highly cited RNA Integrity Number(RIN) of the Agilent 2100 Bioanalyzer system. A high RINe
indicates highly intact RNA, and a low RINe a strongly
degraded RNA sample. In addition, the RNA ScreenTape
assay returns quantitative information as well as theribosomal ratio of eukaryotic and prokaryotic samples.
Choose between the RNA ScreenTape and the HighSensitivity RNA ScreenTape assay depending on therequirements of your application. The High Sensitivity
RNA ScreenTape assay allows a detection of RNA
samples down to 200 pg/μL with a sample volume needof only 2 μL.

41

Next generation sequencing
Impact of gDNA Integrity on the Outcome of DNA Methylation Studies

7

FFPE
[bp]

6
No. of cytosines in millions

1,500

200

5
4
3
2
1

25

0

Initial DIN

4.0

5.8

7.3

7.1

8.3

Conc. (ng/µL)

0.37

0.68

2.29

3.42

2.42

Assay:

4.0

5.8

7.3
DIN

7.1

8.3

Genomic DNA ScreenTape Assay

Abstract:
Human brain tissue from a single donor was used for the genome-wide DNA
methylation analysis. Genomic DNA was extracted from fresh frozen tissue and used as control.
Four tissue samples were subjected to FFPE using varying lengths of paraffin treatment times
to generate varying levels of DNA degradation. The gDNA integrity of this starting material was
determined using the Genomic DNA ScreenTape assay and the DNA Integrity Number (DIN).
To investigate the correlation between DIN and the quality of the sequencing results, the numberof covered CpG sites was determined and compared to the DIN of the initial gDNA samples.
The bar chart demonstrates that the total number of covered CpG sites varies, depending on initial
DIN value of the gDNA samples. The DIN can be used as quality criteria to determine how to handle
individual gDNA samples for downstream workflows, and to ensure successful DNA methylation
analysis.

Application note: 5991-6427EN
42Next generation sequencing
Analysis of DNA from FFPE tissue

600
bp

1

2

3

4

5

6

Sample intensity (FU)

500

400
48,50015,000
7,000
4,000
3,000
2,500
2,000
1,500
1,200
900

300

200

600

100

400
250
100

Assay:

600
900
1,200
1,500
2,000
2,500
3,000
4,000
7,000
15,00048,500

400

250

100

0

Size (bp)

DIN DIN DIN DIN DIN DIN
4.1 5.2 5.0 2.2 2.9 1.9Genomic DNA ScreenTape Assay

Abstract:
Sequencing of genomic DNA (gDNA) from FFPE archived tissue can bechallenging, as the obtained material is often of variable quality. The DNA Integrity Number (DIN)
obtained by the quality control of gDNA using the Agilent Genomic DNA ScreenTape assay has
allowed for a pronounced saving of sequencing and sample preparation overhead. Out of a totalof 751 FFPE samples, a subset of 197 were tested for a correlation of various NGS parametersagainst the DIN. A correlation was identified between DIN and the key parameters of on-target rateand coverage at 10x. The DIN correlates with key sequencing quality metrics and, thus, presents an
integrity threshold for the processing of FFPE samples.

Application note: 5991-5360EN
43

Next generation sequencing
Quality Control of DNA from FFPE tumor samplesA[bp]

B4 C4 D4 E4 F4 G4 H4 A5 B5 C5 D5 E5 F5 G5 H548,50015,000
7,000
4,000
3,000
2,500
2,000
1,500
1,200
900
600
400
250
100

DIN DIN DIN DIN DIN DIN DIN DIN DIN DIN DIN DIN DIN DIN DIN
2.9 4.9 3.9 3.1 2.6 1.5 4.4 4.4 3.2 3.6 2.5 2.3 3.3 3.7 3.5

B500

Sample intensity [FU]

400

300

200

100

Assay:

600

900
1,200
1,500
2,000
2,500
3,000
4,000
7,000
15,00048,500

400

250

100

0
Size (bp)Genomic DNA ScreenTape Assay

Abstract:
To determine if samples were suitable for NGS library preparation, a quality control(QC) was performed at the beginning for a batch of 88 genomic DNA (gDNA) samples from FFPE tumor
tissue by the German Cancer Research Center (DKFZ) High Throughput Sequencing Unit. This initial QC
includes quantitation and analysis with a 4200 TapeStation system and the Genomic DNA assay
to determine DNA quality based on the DNA integrity number (DIN).
The figure shows a representative subset of the data collected on the 4200 TapeStation system with
the Genomic DNA ScreenTape assay. gDNA samples extracted from FPPE material are often of low
DNA integrity, but can still be sufficiently intact for whole exome library preparation and successful
sequencing.

Application note: 5991-7615EN
44

Next generation sequencing
Quantitation of amplified SureSelectQXT whole genome libraries
Agilent 4200 TapeStation
Agilent 2100 Bioanalyzer
2,500

Molarity (pmol/L)

2,000

1,500

1,000

500

0

20 ng Male DNA

50 ng Male DNA

Average size (bp)Starting material
20 ng

50 ng

80 ng

Assay:

80 ng Male DNA
Region molarity (pmol/L)

Agilent 4200
Agilent 2100
Agilent 4200
Agilent 2100
TapeStation System Bioanalyzer System TapeStation System Bioanalyzer System

mean

519

533

850

817

% CV

1.2

2.8

5.4

0.6

mean

849

868

1513

1473

% CV

0.6

1.4

1.7

4.5

mean

1065

1157

2073

2097

% CV

3.8

1.1

4.3

5.5

High Sensitivity D5000 ScreenTape Assay

Abstract:
For multiplex sequencing, SureSelectQXT whole genome libraries are combinedsuch that each index-tagged sample is present in equimolar amounts in the pool. The 4200
TapeStation and 2100 Bioanalyzer systems provide molarity quantitation data along with the sizing
information in the region table of the software. For each library generated by various genomic DNA(gDNA) input amounts, the molarity was plotted in a graph comparing both systems as presented
in the above figure. The data summarized in the table demonstrates that sizing and quantitation of
amplified libraries with the High Sensitivity D5000 ScreenTape assay match the results of the HighSensitivity DNA assay of the 2100 Bioanalyzer system and results can be considered as equivalent.

Application note: 5991-8191EN
45

Next generation sequencing
Quality Control of final NGS libraries

A

B
Lower
L3 (L) G4 H4 A5 B5 C5 D5 E5 F5 G5 H5 A6 B6 C6 D6 E6

100

Upper

4,000
3,000
2,000
1,000

50

C

700

1,000
1,500

400
500

300

200

100

50

25

0

25

Size (bp)

D
30

350

25

300
250

20

Size (bp)Concentration (ng/µL)

268

5,000

Sample intensity [FU]

[bp]

1,500
1,000
700
500
400
300
200

15
10

Assay:

150
100

5
0

200

50
0
0

20

40

60

Sample

80

100

0

20

40

60

80

100

SampleD1000 ScreenTape Assay

Abstract:
The 4200 TapeStation system was used for the quality control of the final NGS
libraries. These were expected to be sized between 250 and 350 bp with a minimum concentrationof 2 ng/μL.
A) Gel view of 15 samples, lane B5 and D6 show negative controls. B) Example of an electropherogramof one sample. The Agilent D1000 assay includes a lower and upper marker. C) The distributionof the concentration for all 80 samples plus eight controls. The two positive controls are shownas green symbols. The red lines indicate the recommended concentration threshold (2 ng/μL).
D) The size distribution of all 80 samples plus eight controls. The two positive controls are shownas green symbols. The red lines indicate the recommend size range (250 to 350 bp).
The analysis of the final NGS libraries with the 4200 TapeStation system confirmed successful DNA
library preparation for all 80 samples and the six positive control samples.

Application note: 5991-7615EN
46

Next generation sequencing
Analysis of NGS Libraries
Equivalence of the 4200 TapeStation and 2200 TapeStation systems
Marker500

Sample intensity (FU)400

Marker300
DNA sample C
200
1:25
1:30

700

1,000
1,500

500

300

200

100

0

50

25

0

400

1:36

100

Size [bp]700600Agilent 2200 TapeStation
concentration [pg/µL]

500

Size [bp]DNA sample C
DNA sample D

Agilent 4200
TapeStation system
Agilent 2200
TapeStation system

400
300
200
100
0

1:25

1:30

900
800

y = 1.0344x
R2= 0.9832

700
600
500
500

600

700

800

900 1,000

1:36

DNA sample C

Assay:

1,000

Agilent 4200 TapeStation concentration [pg/µL]

D1000 and High Sensitivity D1000 ScreenTape Assay

Abstract:
Quality control of NGS libraries is the key for the success of any sequencing run.
The D1000 and High Sensitivity ScreenTape assays can be used for quality control, providing
DNA sizing, and quantitation. The bar chart shows the sizing results of the same sample set
analyzed with the High Sensitivity D1000 ScreenTape assay and the 2200 and 4200 TapeStation
platform. DNA concentration determined with the 4200 TapeStation system is plotted against
the concentration measured with the 2200 TapeStation system for the High Sensitivity D1000
ScreenTape assay.
The data demonstrates that results obtained with the Agilent High Sensitivity D1000 assay,using both TapeStation systems are directly comparable and highly reproducible.

Application note: 5991-6892EN
47

PCR product analysis
Quality Control of Single Cell DNA Samples

Size
[bp]

Measured size [bp]

Upper

1,353

Lower

10,000

2,500

1,353

15

1,500

1,000
1,000

10,000

15

2,000

1,500

Size [bp]Assay:

2,000

2,500

Expected size [bp]D5000 and High Sensitivity D5000 ScreenTape Assay

Abstract:
With current methodologies, both the genome and the whole cell transcript content
can be measured and characterized from a single cell. These analyses require amplification of single
cell starting material for detection. Most-commonly polymerase chain reaction (PCR) is used for thispurpose. To limit the errors introduced by PCR, specific high-fidelity enzymes are required. In addition,
the number of PCR cycles should be kept to a minimum. The Agilent D5000 and Agilent High Sensitivity
D5000 ScreenTape assay can be used for quality control screening of limited-cycle nested PCR amplificationof multiple low-abundance genomic targets from single cells for polymorphic analysis by NGS.
The figure shows a characteristic gel image and electropherogram of one of the amplicons analyzed
using the D5000 ScreenTape assay which was nondetectable by ethidium bromide stained agarose
gels. The right plot displays the correlation between expected amplicon size and amplicon size
measured with the Agilent D5000 ScreenTape assay.
Both TapeStation assays provide unique sizing accuracy and sensitivity, making the detection of
amplicons from low-abundance targets from single cells much easier as compared to traditional
agarose gel-electrophoresis.

Application note: 5991-5259EN
48

Genomic DNA Analysis
Optimization of gDNA Extraction from FFPE tissue500

bp A1 (L)

3h

o/n

DIN
6.0

DIN
6.6

DNA extraction kit A

Sample intensity

400

o/n lysis
DIN 6.6

48,50015,000
7,000
4,000
3,000
2,500
2,000
1,500
1,200
900

300

200

600

3 hour lysis
DIN 6.0

100

400
250

40

600

250

400

Size (bp)10

Spleen

9

35
30

7

25
20

6
5
4

15
10
5
0
A3h

Assay:

Spleen8

DIN

DNA concentration (ng/µL)

100

0

900
1,200
1,500
2,000
2,500
3,000
4,000
7,000
15,00048,500

100

A o/n

B1h

B o/n

C1h

Quant.
limit

3

DIN
functional
limit

1

C o/n

2
0
A3h

A o/n

B1h

B o/n

C1h

C o/n

Genomic DNA ScreenTape Assay

Abstract:
One of the most widely used methods for tissue preservation and archiving is the
preparation of formalin-fixed paraffin-embedded (FFPE) samples. These FFPE tissue archives represent
a valuable source for retrospective studies of gene expression and mutation analysis. However, DNA
extraction from FFPE samples has been proven to be challenging. To compare the effect of DNA
extraction method and the influence of tissue type on the quantity and quality of the extracted DNA,
different mouse tissues isolated by three commercially available DNA extraction kits (vendor A, B andC) were investigated. The electropherogram and the gel image shown in the figure above summarize
the results obtained for the DNA extracted from mouse spleen FFPE tissue for one extraction kit.
The Genomic DNA ScreenTape assay and the DNA Integrity Number (DIN) provide a valuable andreliable tool for quality control of DNA extraction, with quantitation determination and automatedsample integrity assessment. The DIN is presented automatically, and, thus, does not require a
subjective integrity estimation or approximation based on user experience.

Application note: 5991-5246EN
49

Genomic DNA Analysis
Equivalence of the 4200 TapeStation and 2200 TapeStation systems
Agilent 4200 TapeStation system[bp]

gDNA sample A
no dil. 1:2
1:4

Agilent 2200 TapeStation system

gDNA sample B
no dil. 1:2
1:4

[bp]

48,50015,000
7,000
4,000
3,000
2,500
2,000
1,500
1,200
900
600
400
250

gDNA sample A
no dil. 1:2
1:4

gDNA sample B
no dil. 1:2
1:4

DIN
9.4

DIN
8.8

48,50015,000
7,000
4,000
3,000
2,500
2,000
1,500
1,200
900
600
400
250

100

100

DIN
9.1

DIN
9.5

DIN
9.5

DIN
8.7

DIN
8.6

DIN
9.4

10

DIN
9.5

DIN
9.7

DIN
8.6

DIN
9.6

Agilent 4200
TapeStation system
Agilent 2200
TapeStation system

9
8
7

DIN

6
5
4
3
2
1
0

Assay:

no dil.
1:2
1:4
gDNA sample A

no dil.
1:2
1:4
gDNA sample B

Genomic DNA ScreenTape Assay

Abstract:
To evaluate the difference between the 2200 TapeStation system and the 4200
TapeStation system, two commercially available gDNA samples were analyzed at three different
concentrations using the Genomic DNA ScreenTape assay. The DNA Integrity Number (DIN) is
automatically determined and directly displayed below the individual lane of the gel image. A DIN
is calculated on a scale from 1 to 10. A high DIN indicates highly intact gDNA, whereas a low DIN
corresponds to a strongly degraded gDNA. Both gDNA samples were highly intact, as indicated by
the determined DIN which is directly displayed below the gel image.
The bar chart illustrates that the DNA integrity analysis with the 4200 TapeStation system is highly
comparable to that of the 2200 TapeStation system. The precision for the DNA integrity analysis
for both TapeStation systems is 4 % for all tested samples and concentrations.

Application note: 5991-6892EN
50

Genomic DNA Analysis
Quantitation of isolated genomic DNA (gDNA) samples

Sample concentration of extracted genomic DNA as
determined by three different quantification platforms
70.0

Concentration [ng/µL]

60.0
50.0
40.0

NanoDrop
Qubit

30.0

TapeStation
20.0
Error bars = S.Error
10.0
0.0
Protocol A

Assay:

Protocol B

Protocol C

Agilent

Genomic DNA ScreenTape Assay

Abstract:
The Genomic DNA ScreenTape assay in combination with the TapeStation systemallows the automatic analysis of up to 96 genomic DNA samples. With the DNA Integrity number(DIN) the assay provides automatic determination of sample integrity as well as sample quantitation.
In the above example, genomic DNA from HEK293 cells was isolated with extraction kits fromdifferent suppliers. Samples were analyzed as triplicates and the quantitation data was collected foreach kit. The samples were also quantified using the Qubit dsDNA broad range assay and NanoDrop
spectrophotometer to compare results. The mean values obtained from the Qubit, NanoDrop, and the
Genomic DNA ScreenTape assay were plotted, compared, and shown above.
The data shows that the ScreenTape and Qubit analysis provide very similar results with a minimum
average difference of 1 % and a maximum average difference of 5 %. The CV percentages are similar
across all three platforms for each kit illustrating differences between each triplicate extracted
sample rather than any performance variances in the platforms.

Application note: 5991-1797EN
51

Genomic DNA Analysis
Quantity and Integrity Analysis of genomic DNA for Next Generation
Sequencing

[bp]

70

B1

C1

DIN
9.2

DIN
7.9

DIN
8.3

Qubit
Agilent 4200 TapeStation
Nanodrop60

48,50015,000
7,000
4,000
3,000
2,500
2,000
1,500
1,200
900
600
400

50
Concentration [ng/µL]

A1

40
30
20

250

10

100

0
First dilution

Assay:

Second dilution

Genomic DNA ScreenTape Assay

Abstract:
The Agilent SureSelectQXT WGS protocol requires high-quality DNA samples for
optimal performance and precise quantitation of the gDNA starting material. Serial quantitation
was carried out using the Qubit instrument with the dsDNA BR Assay in accordance to the protocol.
The same samples were analyzed on the Genomic DNA ScreenTape assay with the 4200 TapeStation
system and the NanoDrop with six replicates on each instrument. Data from the 4200 TapeStation
system, Qubit, and NanoDrop are presented in the above figure, showing the applicability of the
Genomic DNA ScreenTape assay in quantitating genomic DNA starting material. As shown in theabove bar chart, quantitation results of the 4200 TapeStation and the Qubit instrument are in good
agreement. The measurement of genomic DNA with UV spectroscopy tends to overestimate thequantity due to other buffer components that may absorb in the UV spectrum.
In addition, the Genomic DNA ScreenTape assay provides objective assessment of sample integrity
within the same QC step. Sample integrity is automatically determined by the DNA Integrity Number(DIN) calculation provided by the TapeStation analysis software.

Application note: 5991-8191EN
52

Genomic DNA Analysis
Integrity analysis of Genomic DNA from bacterial sources

53,929Lower

300
DIN 9.2

Sample intensity (FU)

600
500
400
300
200
100

200
150
100
50

600

900
1,200
1,500
2,000
2,500
3,000
4,000

600

900
1,200
1,500
2,000
2,500
3,000
4,000

7,000
15,00048,500

400
400

100

7,000
15,00048,500

250

1,106

Lower

6,894

DIN 3.0

Sample intensity (FU)

300
250
200
150
100
50

DIN 1.1
400
300
200
100
0
100

900
1,200
1,500
2,000
2,500
3,000
4,000
7,000
15,00048,500

600

400

100

250

0

7,000
15,00048,500Sample intensity (FU)

900
1,200
1,500
2,000
2,500
3,000
4,000

600

400

100

250

Lower

Assay:

250

0

0

350

DIN 6.2

250

Sample intensity (FU)700

10,030Lower

800

Genomic DNA ScreenTape Assay

Abstract:
The DNA Integrity Number (DIN) was developed to facilitate the quality control of
isolated genomic DNA (gDNA) samples. The DIN software algorithm allows for the classificationof total DNA based on a numbering system from 1 to 10, with 1 being the most degraded and 10
being the most intact. The DIN facilitates the interpretation of electropherograms, allows for thecomparison of samples, and ensures the repeatability of experiments and quantitation of high-quality
gDNA moving into e.g. next generation sequencing library construction.
The above electropherograms illustrate the variability of the analyzed genomic DNA samples isolated
from bacteria. DIN numbers range from 9.2 down to 1.1. In earlier studies, it was determined thatsamples with a DIN of > 7 were acceptable to progress into the next step of library construction.
In the example above, 3 out of 4 samples did not pass the QC criteria for the downstream application.

Application note: 5991-5442EN
53

Gene Expression Analysis

C1

D1

T = 0 min

E1

F1

G1

H1

T = 5 min

A2

B2

A

T = 10 min

×10 3
5Sample intensity [FU][nt] A1 (L) B1

6,000
4,000
2,000
1,000
500
200

4

2

FLuorescence (dRn)

3

1,000

2,000

25

200

YWHAZ

HPRT1
5' AssayHigh intact
Medium intact
Degraded
NRT
NTC

3' Assay

5' Assay

3' Assay

0.0
-1.0
-2.0
-3.0
-4.0

1

Assay:

Relative Cqdifference between high integrity RNA versus
medium integrity and degraded RNA

Amplification plots HPRT1-5’RNA degradation

2

0
0
-1

500

0

B

4

Size [nt]3

RINe RINe RINe RINe RINe RINe RINe RINe RINe
9.8 9.8 9.8 4.5 4.5 4.6 2.3 2.2 2.2

5

T = 0 mins
T = 5 mins
T = 10 mins

1

25

6

B1
E1
H1

4,000
6,000

Impact of RNA degradation on qPCR experiments

-5.0
10

20
Cycles

30

40

-6.0
Medium integrity
Degraded

RNA ScreenTape Assay

Abstract:
The importance of using only high integrity RNA samples for real-time quantitative
PCR (RT-qPCR) experiments as recommended by MIQE (Minimum Information for Publication of
Quantitative Real-Time PCR Experiments) guidelines was shown by carrying out RT-qPCR amplification
using heat-degraded RNA as a template. The differentially heat-degraded RNA and untreated RNA wereanalyzed using the 2200 TapeStation and 2100 Bioanalyzer systems and designated as high, medium,and low integrity based on the RNA integrity number equivalent (RINe) The gel image as well as theelectropherogram trace overlay shows RNA with three different levels of degradation.
qPCR amplifications were carried out using different integrities of total RNA. The amplification plots
are shown in the lower left figure. The Cq was determined for all primer pairs. The Cq value of theuntreated and highly intact RNA sample was taken as a reference. The relative Cq difference of thetreated RNA templates was calculated, and is presented in the lower right figure. These results showthat differential degradation is specific to the respective gene products and that the RNA ScreenTape
assay can be reliably used in determining the integrity of total RNA samples. The TapeStation systemprovides an ideal system to fit into any qPCR workflow in accordance with the MIQE guidelines.

Application note: 5991-4971EN
54

Analysis of total RNA
Equivalence of the 4200 TapeStation and 2200 TapeStation system
RNA integrity number

×103
16

Agilent RNA ScreenTape assay
10

12

9
8

10

7
7

6

6

RINe

Sample intensity (FU)14

Liver total RNA
1:8
1:20

Marker

4

4
3

1:35

2

2

1
Size [nt]

4,000
6,000

2,000

1,000

200

500

0
0

25

0

5

1:8

1:20

1:35

Agilent 4200 TapeStation system
Agilent 2200 TapeStation system

Assay:RNA and High Sensitivity RNA ScreenTape Assay

Abstract:
As RNA is highly sensitive to degradation, quality control is essential, and usually
includes RNA integrity analysis and quantitation. The above electropherogram overlay represents
different dilutions of liver total RNA analyzed on the 4200 TapeStation system and the RNA
ScreenTape assay.
The RNA Integrity Number equivalent (RINe), indicating RNA integrity, is automatically determinedby the TapeStation Software. The RINe is calculated on a scale from 1 to 10. A high RINe indicateshighly intact total RNA, whereas a low RINe corresponds to a strongly degraded sample. The bar
chart illustrates that RNA integrity analysis is highly reproducible and independent of the analyzed
concentration for both TapeStation systems. In comparison to the 2200 TapeStation system, the4200 TapeStation system further reduces hands-on-time, allowing the analysis of 96 samples
without manual intervention. This is a primary requirement specifically in a high throughput
environment.

Application note: 5991-6892EN
55

Analysis of total RNA
Equivalence of the 4200 TapeStation and 2200 TapeStation system
quantitation

Agilent RNA ScreenTape assay

Agilent High Sensitivity RNA ScreenTape assay

200

Concentration [pg/µL]

Concentration [ng/µL]

20,000
150

100

50

15,00010,000

5,000

0

0
1:8

1:20

1:100

1:35

1:200

1:500

Agilent 4200 TapeStation system
Agilent 2200 TapeStation system

Assay:RNA and High Sensitivity RNA ScreenTape Assay

Abstract:
Liver total RNA of various concentrations was quantified with the Agilent RNA
ScreenTape assay and the Agilent High Sensitivity RNA ScreenTape assay using the Agilent 4200
TapeStation system and the Agilent 2200 TapeStation system (n=15). The data demonstrates
that the total RNA quantitation with both TapeStation systems is comparable for the 2 assays.
The total RNA quantitation precision with the 4200 TapeStation system was below 15 % CV for
the RNA and High Sensitivity RNA ScreenTape assays.

Application note: 5991-6892EN
56

Analysis of total RNA
Comparison of the RNA integrity analysis on 2200 TapeStation with 2100
Bioanalyzer system

RNA source organism

RNA source organism
10

Human
Mouse
Rat

9
8

8

7

7

6

6

5

5

4

4

3

3

2

2

1

1
1

Assay:

2

E. coli
R. solanacearum

9

RINe

RINe

10

3

4

5
6
RIN

7

8

9

1

10

2

3

4

5

6

7

8

9

10

RIN

RNA ScreenTape Assay

Abstract:
Agilent pioneered the reliable and robust assessment of total RNA quality with theintroduction of the RNA Integrity Number (RIN) for the 2100 Bioanalyzer system. Despite differencesin the technologies between the two systems, the 2200 TapeStation system provides the same quality
assessment with the RNA integrity number equivalent (RINe).
To demonstrate that RIN and RINe are directly comparable, various RNA samples were analyzed on boththe 2100 Bioanalyzer and the 2200 TapeStation systems. RINe and RIN, values obtained for eukaryotic
total RNA were plotted against each other (left figure). The median error calculated versus the 2100
Bioanalyzer system was a 0.4 RIN unit difference, and a standard deviation of 0.28 RIN units.
A similar approach was taken to compare the obtained RINe and RIN values from prokaryotic total RNA(right figure). Similar results were obtained for the prokaryotic RNA with a median error of 0.2 RIN units
and a standard deviation over dilution series of 0.16 RIN units.

Technical Overview: 5991-3426EN
57

Analysis of total RNA
Equivalence of RNA analysis on 2200 TapeStation and 2100 Bioanalyzer
systemsA

B
A1 (L)

B1

C1

D1

E1

F1

G1

Total
Ladder RNA

Total
RNA

Total
RNA

Total
RNA

Total
RNA

Total
RNA

RIN
9.6

RIN
9.6

RIN
9.7

RIN
9.5

RIN
9.7

RIN
9.6

4,000

6,000
4,000
2,000

2,000

1,000

1,000

500
500

200

200
25

25
RINe
9.4

RINe
9.5

RINe
9.4

RINe
9.6

RINe
9.4

RINe
9.5

Integrity (RINe/RIN)

Quantitation (ng/µL)
SystemAgilent 2200
TapeStation

Agilent 2100
Bioanalyzer

Agilent 2200
TapeStation

Agilent 2100
Bioanalyzer

Average

128.8

116.5

9.5

9.56

CV %

2.8

7.4

0.9

0.8

Assay:RNA ScreenTape Assay

Abstract:
Quality control of total RNA to determine its integrity is essential to ensure thathigh quality starting material is used for e.g. RNA-Seq library preparation. The total RNA isolated fromthe HEK293 cell lines was analyzed using the RNA ScreenTape and RNA 6000 Nano assays on the2200 TapeStation and 2100 Bioanalyzer systems, respectively. The SureSelect protocol indicatesthat a RNA integrity number (RIN) of 8 or above should be used for RNA library preparation. Gel-like
images generated by the analysis software from both instruments are shown in the above figure
together with calculated RINe and RIN values with an average of 9.5. The table summarizes the RNA
integrity and quantity metrics obtained from both instruments, and shows that the 2200 TapeStation
system is highly comparable to the 2100 Bioanalyzer system for quantitation and analyzing the
integrity of total RNA.

Application note: 5991-4116EN
58

RNA Quantitation

600

RNA
NanoDrop

500
400
300
200
100
0
500

200

100

50

25

Reported concentration [ng/µL]

Reported concentration [ng/µL]

RNA Quantitation with the TapeStation system in comparison with
spectrophotometric and fluorescence based measurements

×103
14

HS RNA
Qubit

12
10
8
6
4
2
0
10,000

Assay:

6,250

2,500

1,000

500

Nominal concentration [pg/µL]

Nominal concentration [ng/µL]

RNA and High Sensitivity RNA ScreenTape Assay

Abstract:
To determine the quantitation accuracy and reproducibility of the RNA ScreenTape
assay, rat kidney total RNA samples were prepared at five different nominal concentrations ranging
from 25 to 500 ng/μL. The same set of samples was analyzed with the RNA ScreenTape assay on
the 2200 TapeStation system and the NanoDrop 2000 system by four different analysts (left figure).
Both quantitation methods, the RNA ScreenTape assay and the NanoDrop 2000 system, yieldedcomparable results for the tested concentrations.
Similarly, the quantitation accuracy and reproducibility of the High Sensitivity RNA ScreenTape assay
was tested. Rat kidney total RNA samples were prepared at five different nominal concentrations
ranging from 500 to 10,000 pg/μL. The samples were analyzed with the High Sensitivity RNA
ScreenTape assay on the 2200 TapeStation system and this time the Qubit 2.0 Fluorometer wasused by two different analysts (right figure). Both quantitation methods, the High Sensitivity RNA
ScreenTape assay and the Qubit 2.0 Fluorometer, yielded comparable results for the tested samples.
The data generated confirms that quantitation results for both RNA ScreenTape assays are in good
agreement with data generated with spectrophotometric and fluorometric methods.

Technical Overview: 5991-3426EN
59RNA Purity
Detection of genomic DNA contaminationA

BeforeAfter

3

DNAse ×10
5

Sample intensity (FU)

DNAse

6,000
4,000
2,000
1,000
500

B
Lower

4

3
28S

2

18S

200

1
25

NanoDrop

6,000

4,000

2,000

1,000

500

Size [nt]Agilent 2200 TapeStation system

Total conc. RNA(ng/µL)
purity (%)

Assay:

25

RINe
8.5

200

0
RINe
8.8

Total conc. RNA region gDNA region RNA
Purity(ng/µL)conc. (ng/µL) conc. (ng/µL) purity (%) accuracy (%)

290

69.0

276

175

98

63.4

275

72.7

321

221

95

68.8

91.9
94.7

250

80.0

246

177

64

72.0

89.9

225

88.9

237

198

37

83.5

94.0

210

95.2

194

175

16

90.2

94.7

RNA ScreenTape Assay

Abstract:
During RNA purification procedures, residual genomic DNA can be present. This canlead to inaccurate RNA quantitation or cause issues with downstream applications. The identificationof genomic DNA contamination can therefore be useful in deciding whether further cleanup of theextracted RNA is required. In contrast to some capillary based systems, the 2200 TapeStation system
can resolve intact genomic DNA contaminants from the large ribosomal RNA. The figure shows theanalysis of rat kidney total RNA spiked with mouse genomic DNA. The genomic DNA contamination
results in an additional peak running above the 28S RNA peak. Treatment of the RNA sample with
DNase shows that this spiked DNA is removed.

Technical Overview: 5991-3426EN
60

Analysis of FFPE RNA
DV200 Evaluation with RNA ScreenTape Assays

Assay:RNA and High Sensitivity RNA ScreenTape Assay

Abstract:
The DV200 quality metric represents the percentage of RNA fragments above 200
nucleotides and shows a high correlation to the precapture library yield of RNA samples originating fromformalin-fixed paraffin-embedded (FFPE) tissue. The RNA and High Sensitivity RNA ScreenTape assays
enables fast and easy analysis of FFPE RNA samples, with the TapeStation Analysis software displaying
DV200 results after region setup as percentage of total. DV200 region setup can be automated for
repeated FFPE RNA sample analysis and all region data can be exported and reported. Both the RNA
ScreenTape assay and High Sensitivity RNA ScreenTape assay yielded highly comparable results.

Application note: 5991-8355EN
61

LIMS Integration
Integration of the 4200 TapeStation system into a laboratory information
management system (LIMS)
Fast-track sample
and ready tosequence
multiplexes
LIMS
Sample ID

Platelayout/ID

Copy/paste

Scan plate ID

Agilent 4200
TapeStation Run
Controller
SoftwareAgilent 4200
TapeStation .xml
Analysis
Software

Dedicated
network
drive

Abstract:
Large core sequencing facilities implement a laboratory information management
system (LIMS) to handle all sample relevant data, from pre-analytical information e.g. sample
IDs and sample source down to analytical parameters like sample concentration, molarities andsequencing results. The 4200 TapeStation software package allows a seamless LIMS integration.
The 4200 TapeStation controller software offers different methods for data import from a database.
Sample IDs can be imported from csv-files and additional information like plate ID or lot informationof reagents and ScreenTape devices can be entered into the software with an external handheld
barcode scanner. Results data can be exported as an .xml file and saved on a dedicated network
drive. The .xml-file contains all relevant result data like sizing, concentration, and integrity information
for analyzed DNA and RNA samples as well as peak sizes, areas, heights, molarities, and region
specific information.
The 4200 TapeStation system offers full automation of RNA and DNA sample quality control and areduction in hands-on work. In addition, LIMS integration further reduces manual data entry steps
resulting in increased efficiency and elimination of a potential source of errors.

Technical Overview: 5991-7984EN
62

Literature
Application notes and other publications
To download an application note or to find other literature on the2100 Bioanalyzer and 4200 Tapestation systems visit our websites:www.agilent.com/genomics/bioanalyzer andwww.agilent.com/genomics/tapestation

Application notes 2100 Bioanalyzer system
DNA analysis

Publication Number

Next-generation sequencing
Improving sample quality for SureSelect target enrichment andnext-generation sequencing with the High Sensitivity DNA kit
Performance characteristics of the High Sensitivity DNA kit for the
Agilent 2100 Bioanalyzer
DNA quality control of formalin-fixed paraffin-embedded and fresh-frozen
tissues prior to target-enrichment and next generation sequencing
Low input DNA size selection on the Pippen Prep System using
the Agilent 2100 Bioanalyzer system
PCR product analysis
Optimizing real-time quantitative PCR experiments with theAgilent 2100 Bioanalyzer
Food analysis
Use of the Agilent 2100 Bioanalyzer for basmati rice authenticity
Strawberry and raspberry fruit differentiation using theAgilent 2100 Bioanalyzer
Identification of different meat species by the Agilent Fish ID solution
on the Agilent 2100 Bioanalyzer

5990-5008EN
5990-4417EN
5990-0483EN
5990-8382EN

5989-7730EN
5989-6836EN
5990-3327EN
5990-8452EN

RNA analysis
Analysis of total RNA
RNA Integrity Number (RIN) - Standardization of RNA quality control
Simplified DV200 Evaluation with the 2100 Bioanalyzer System
Assessing integrity of plant RNA with the Agilent 2100 Bioanalyzer
Assessing Integrity of Insect RNA

635989-1165EN
5991-8287EN
5990-8850EN
5991-7903EN

Analysis of Small RNA
Analysis of miRNA content in total RNA preparations using theAgilent 2100 Bioanalyzer
Agilent integrated solutions for design, synthesis, and quality controlof guide RNA for CRISPR-Cas9 genome editing workflows

5989-7870EN
5989-8539EN

Genome Editing
Analysis of total RNA
Agilent integrated solutions for design, synthesis, and quality controlof guide RNA for CRISPR-Cas9 genome editing workflows

5991-7557EN

Protein analysis
Protein purification
Monitoring protein fate during purification with theAgilent 2100 Bioanalyzer

5990-6153EN

Analysis of modified proteins
Analysis of PEGylated proteins using the Agilent 2100 Bioanalyzer

5990-9593EN

High sensitivity protein analysis
Performance characteristics of the High Sensitivity Protein 250 assay
Immunoprecipitation and the High Sensitivity Protein 250 Assay
Quantification strategies with the High Sensitivity Protein 250 assay

5989-8940EN
5990-4097EN
5989-8941EN

Antibody analysis
Protein analysis with the Agilent 2100 Bioanalyzer–
An overview of the protein kit portfolio

5990-5283EN

Food analysis
Rapid wheat varietal identification using the Agilent 2100 Bioanalyzer
and automated pattern-matching
Milk protein analysis with the Agilent 2100 Bioanalyzer and theAgilent Protein 80 kit

645989-7735EN
5990-8125EN

Application notes 2200 and 4200 TapeStation systems
DNA analysis

Publication Number

Next-generation sequencing
Impact of gDNA Integrity on the Outcome of DNA Methylation Studies
5991-6427EN
The DNA Integrity Number (DIN) Provided by the Genomic DNA ScreenTape
Assay Allows for Streamlining of NGS on FFPE Tissue Samples
5991-5360EN
Use of the Agilent 4200 TapeStation System for Sample Quality Control
in the Whole Exome Sequencing workflow at the German Cancer
Research Center (DKFZ)
5991-7615EN
QXT
Quality Control for Agilent SureSelect WGS Library Preparation
5991-8191EN
Evaluating the Agilent 4200 TapeStation System for High Throughput
Sequencing Quality Control
5991-6892EN
PCR product analysis
Quality Control of Single Cell DNA Samples with the Agilent D5000
ScreenTape Assays for the Agilent 2200 TapeStation System
Genomic DNA
The DNA Integrity Number (DIN) Provided by the Agilent 2200 TapeStation
System is an Ideal Tool to Optimize FFPE Extraction
Evaluating the Agilent 4200 TapeStation System for High Throughput
Sequencing Quality Control
Analysis of high molecular weight genomic DNA using the Agilent 2200
TapeStation and Genomic DNA ScreenTape
Quality Control for Agilent SureSelectQXT WGS Library Preparation
Integrating the DNA Integrity Number (DIN) to Assess Genomic
DNA (gDNA) Quality Control Using the Agilent 2200 TapeStation System655991-5259EN

5991-5246EN
5991-6892EN
5991-1797EN
5991-8191EN
5991-5442EN

RNA analysis
Gene Expression Analysis
A Systematic Approach to Optimize Real-Time Quantitative RT-qPCR
Experiments with the Agilent 2200 TapeStation System
Analysis of total RNA
Evaluating the Agilent 4200 TapeStation System for High Throughput
Sequencing Quality Control
Performance of the Agilent RNA ScreenTape and the High Sensitivity RNA
ScreenTape Assay for the Agilent 2200 TapeStation System
Quality Control for SureSelect Strand-Specific RNA Library Preparation
Using the Agilent 2200 TapeStation System5991-4971EN

5991-6892EN
5991-3426EN
5991-4116EN

RNA Quantitation
Performance of the Agilent RNA ScreenTape and the High Sensitivity RNA
ScreenTape Assay for the Agilent 2200 TapeStation System5991-3426EN

RNA Purity
Performance of the Agilent RNA ScreenTape and the High Sensitivity RNA
ScreenTape Assay for the Agilent 2200 TapeStation System5991-3426EN

Analysis of FFPE RNA
DV200 Evaluation with RNA ScreenTape Assays

5991-8355EN

LIMS integration
LIMS integration of the Agilent 4200 TapeStation System665991-7984EN

Applications for DNA, RNA, and Protein Analysis
This compendium highlights key applications using the Agilent 2100 Bioanalyzer and4200 TapeStation systems.
From next generation sequencing, genomic DNA, RNA, genome editing to protein
analyses, this compilation of applications will help you accelerate discovery andsupport your research.

Learn more:

www.agilent.com/genomics/tapestationwww.agilent.com/genomics/bioanalyzer
Buy online:

www.agilent.com/chem/store
U.S. and Canada

1-800-227-9770
agilent_inquiries@agilent.com
Europeinfo_agilent@agilent.com
Asia Pacificinquiry_lsca@agilent.com

For Research Use Only.
Not for use in diagnostic procedures.
This information is subject to change without notice.
© Agilent Technologies, Inc. 2018
Published in the USA, January 20, 20185991-8974EN