Application Note
Genomics

Assessment of Long IVT mRNA
Fragments with the Agilent 5200
Fragment Analyzer system

Authors
Denise Warzak, Chava Pocernich,and Kit-Sum Wong
Agilent Technologies, Inc.

Abstract
The use of in vitro transcription (IVT) mRNA is becoming widespread in researchareas such as ribozyme and aptamer synthesis, mRNA synthesis, RNA interference,
and antisense RNA techniques. Longer RNA transcripts greater than 3,000 nt areneeded for gene structure and functional studies. Reliable sizing, quantification,and quality assessment of IVT mRNA greater than 6,000 nt was obtained using
the Agilent 5200 Fragment Analyzer system with the Agilent RNA kit (15 nt) and an
extended RNA method.

Introduction

Experimental

The synthesis of IVT mRNA results inmany different types of RNA including
viral RNA, mRNA, aptamer, dsRNA,CRISPR gRNA, riboprobe, and miRNA
with endless application possibilities.
Conventionally synthesized RNAfragments are less than 100 nt in length,
but research in gene structure andfunctional studies requires RNA longer
than 3,000 nt. In addition, RNA is proneto degradation due to factors including
structure, light, heat, and RNases. Theability to detect low amounts of RNA
degradation is important to the successof diverse RNA applications. The parallel
capillary electrophoresis instrumentation
portfolio from Agilent Technologies
provides sensitive and reliable solutions
for assessing varied lengths of RNA
constructs1. The Fragment Analyzer
system with the RNA kit (15 nt) provides
reliable sizing and quality assessment ofshort and long IVT RNA.

The experiments in this study wereperformed with a 5200 Fragment
Analyzer system and can be replicatedwith comparable results on Agilent 5300
and 5400 Fragment Analyzer systems.

Separation method of RNA transcripts
IVT mRNA was produced using theRiboMAX Large Scale RNA Production
System (Promega, #P1300). RNAtranscripts 9,000 and 10,000 nt in length
were diluted in nuclease-free water.
The diluted transcripts were separated
on the Agilent 5200 Fragment Analyzer
system with the Agilent RNA kit (15 nt)(p/n DNF‑471) with the followingmodifications.
The Agilent RNA Ladder(p/n DNF‑382-U020) was replaced with
the Lonza RNA marker 500, 1,000, 1,500,2,000, 2,500, 3,000, 4,000, 5,000, 6,000,
and 9,000 nt (Lonza, #50575) (longRNA Ladder). The stock of long RNA
Ladder was diluted in nuclease-freewater to 96 ng/µL, and the concentration
confirmed with the Qubit 4.0 using theRNA HS assay kit (Thermo Fisher Sci.,#Q32852). The long RNA Ladder wasadded to the Agilent RNA Diluent Marker(15 nt) (p/n DNF-369-0004) at the sameratio as described in the RNA kit protocol(2 µL ladder to 22 µL RNA Diluent
Marker).

The standard separation method (8 kVfor 45 minutes) for the RNA kit (15 nt)
was manually altered in the Agilent5200 Fragment Analyzer software(extended RNA method) for fragments
greater than 6,000 nt to improve theresolution of high molecular weightfragments. The extended RNA method
has a lower separation voltage (4 kV)
and longer separation time (90 minutes)
providing better separation of impurities
from fragments greater than 6,000 nt(Table 1).
The extended RNA method with thelong RNA Ladder was employed for all
subsequent runs described within thisApplication Note.

Degradation series
A 100 µL sample of the 9,000 nt IVTmRNA (100 ng/µL) was heat denatured
in a thermocycler at 70 °C. 12 µL aliquots
were collected every two minutes, for
16 minutes, from the PCR tube andplaced on ice until further analysis. The
degradation series was then analyzed
according to the separation method
previously described.

Capped versus uncapped
A 1,800 nt IVT mRNA was transcribedusing the RiboMAX kit (Promega,#P1711) with and without 5’ capping andseparated with the described extended
RNA method.

Table 1. The Agilent RNA kit (15 nt) standard and extended
method protocol for the Agilent 5200 Fragment Analyzer system.
Agilent RNA Kit (15 nt)

2Sample Injection

Separation Method

Standard Method

5 kV, 4 seconds

8 kV, 45 minutes

Extended Method

5 kV, 4 seconds

4 kV, 90 minutes

48

51

54

28

30
32
Time (min)

B

LM

3,000
2,000

34

9,000
36

9,000

26

4,000
5,000
6,000

24

1,500
2,000
2,500
3,000
4,000
5,000
6,000

1,000

500
22

2,000
2,500
3,000

20

1,500

4,000

18

1,000

5,000

LM

500

6,000

A

method displayed enhanced resolution
compared to the standard method asseen by the increased spacing between
the ladder peaks and the increased
sharpness of the 9,000 nt peak. The
enhanced resolution achieved with theextended method aids in the detectionof minor degradation, contamination,or sizing differences from incomplete
transcription.

155,000
4,500
4,000
3,500
3,000
2,500
2,000
1,500
1,000
500
015As IVT mRNA transcripts become longer,
sizing ladders need to be extended
and analysis methods re-evaluated toprovide the best possible results. The
5200 Fragment Analyzer system with
the RNA kit (15 nt) provides accurate
sizing up to 6,000 nt for IVT RNA. To
accommodate accurate sizing of longer
IVT mRNA, a ladder with appropriate
length fragments is required. TheLonza RNA marker (long RNA Ladder)is suitable for sizing IVT mRNA above
6,000 nt due to the additional numberof fragments: 500, 1,000, 1,500, 2,000,2,500, 3,000, 4,000, 5,000, 6,000, and
9,000 nt and was used in place of theAgilent RNA Ladder (15 nt). Longer
fragments require extended separation
time, which aids in achieving the best
resolution possible. The RNA kit (15 nt)
separation method was manuallyadapted in the 5200 Fragment Analyzer
system software (extended RNAmethod) to facilitate separation of IVTmRNA longer than 6,000 nt and provide
better resolution of impurities from thepeak of interest.

RFU

Comparison of the normal andextended RNA method

The long RNA Ladder was diluted
to 96 ng/µL and separated on the5200 Fragment Analyzer systemwith the RNA kit (15 nt) standard
method and the extended RNA method(Figure 1). Both methods provided
similar separation pattern of all
10 ladder fragments. The long RNA
Ladder separated with the extended

RFU

Results and discussion

6972384042818444

1,00004245576063 66Time (min)75788790Figure 1. The long RNA Ladder separated on the Agilent 5200 Fragment Analyzer system with theAgilent RNA kit (15 nt) (A) normal method (8 kV, 45 minutes) (B) extended RNA method (4 kV, 90 minutes).
LM = lower marker.

38,000
7,000A Normal RNA method

6,000
RFU

5,000
4,000

LM

3,000
2,000

1,500
2,000
2,500
3,000
4,000
5,000
6,000

9,000
9,000

1,000500

15Size (bp)Size (bp)

B Extended RNA method

4,000
5,000
6,0001,000

2,000
2,500
3,000

LM500

20,00018,00016,00014,00012,00010,000
8,000
6,000
4,000
2,000
015RFU0

1,500

1,000

Figure 2. Overlay of the 9,000 nt IVT mRNA fragments over a dilution series of 100, 50, 10, 1 ng/µL
separated on the Agilent 5200 Fragment Analyzer system with the Agilent RNA kit (15 nt) using the long
RNA Ladder and (A) the normal RNA method (B) the extended RNA method. LM = lower marker.
5,000
4,500

A Normal RNA method

4,000
3,500
LM

3,000
RFU

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

9,000

Size (bp)9,000500

1,500
2,000
2,500
3,000
4,000
5,000
6,000

14,000

1,000

0

1,500

500
15Sizing with the long RNA Ladder wasassessed with the 9,000 and 10,000 nt
IVT mRNA transcripts. The RNA kit(15 nt) provides reliable quantitation
for a single RNA fragment with aconcentration range from 1 to 100 ng/µL.
To reflect this, a dilution series of eachsample over the entire concentration
range of the kit (100, 50, 10 and 1 ng/µL)
was assessed on the 5200 Fragment
Analyzer system with the RNA kit (15 nt)
standard RNA method (Figure 2) andthe extended RNA method (Figure 3).
Both methods reported precise sizing as
indicated by the low percent coefficientof variance (% CV) throughout thedilution series (Table 2). In addition, alow percent error throughout the dilution
series for both samples and with bothmethods indicated accurate sizing. The
extended RNA method reported a lower
average percent error (–0.7 %) over theentire concentration range comparedto the standard RNA method (8.4 %)for the 9,000 nt sample. The 10,000 nt
IVT mRNA average percent sizing error
was similar between the two separation
methods. The extended RNA method isrecommended when extremely accurate
sizing or high resolution is needed for
determining the presence of degradationor sizing differences from incomplete
transcription in IVT mRNA samples
longer than 6,000 nt.

Size (bp)

B Extended RNA method

12,000RFU

10,000
8,000
6,000
4,000

LM

4,000
5,000
6,000

2,000
2,500
3,000

1,000

500

0

15

2,000

Figure 3. Overlay of the 10,000 nt IVT mRNA fragments over a dilution series of 100, 50, 10, 1 ng/µL
separated on the Agilent 5200 Fragment Analyzer system with the Agilent RNA kit (15 nt) using the long
RNA Ladder and (A) the normal RNA method (B) the extended RNA method. LM = lower marker.

4A

Sizing Throughout Dilution Series
Theoretical Size 8,989 nt

Concentration(ng/µL)

Normal RNA Method

Extended RNA Method

Average* (nt)

% CV

% Error

Average* (nt)

% CV% Error

100

9,910

1.6 %

10.2 %

8,705

0.9 %

-3.2 %

50

9,862

0.8 %

9.7 %

9,051

2.3 %

0.7 %

109,718

0.6 %

8.1 %

8,977

0.4 %

-0.1 %

19,491

0.4 %

5.6 %

8,971

0.7 %

-0.2 %

1 to 100

9,745

0.8 %

8.4 %

8,926

0.8 %

-0.7 %

B

Sizing Throughout Dilution Series
Theoretical Size 10,003 nt

Concentration(ng/µL)

Normal RNA Method

Extended RNA Method

Average* (nt)

% CV

% Error

Average* (nt)

% CV% Error

100

10,736

0.2 %

7.3 %

9,453

1.3 %

-5.5 %

50

10,676

0.3 %

6.7 %

9,501

1.1 %

-5.0 %

10

10,496

0.4 %

4.9 %

9,597

0.8 %

-4.1 %

1

10,197

0.8 %

1.9 %

9,620

0.2 %

-3.8 %

1 to 100

10,526

0.4 %

5.2 %

9,543

0.5 %

-4.6 %

02

468

9,000

B

1,000

LM

1,500
2,000
2,500
3,000
4,000
5,000
6,000

Size(nt)

A500

26,00024,00022,00020,00018,00016,00014,00012,00010,000
8,000
6,000
4,000
2,000
0

15

The smear analysis feature in the Agilent
ProSize data analysis software canbe used to determine the percent ofdegradation from the DNA template, andthe percent of IVT mRNA. The 9,000 nt
IVT mRNA was degraded at 70 °C for
0 to 16 minutes. Every two minutes analiquot was taken and placed on ice. The
degradation series was separated on the5200 Fragment Analyzer system with theRNA kit (15 nt) using the extended RNA
method and long RNA Ladder (Figure 4).
The electropherogram and gel image
displayed a decrease in the expectedpeak height with increased degradation
time. Smear analysis was performed todetermine the percent of degradation(smear range 75 to 7,500 nt) and thepercent of the peak of interest (smearrange 7,500 to 11,000 nt). The percent
concentration was recorded for eachsmear range and compared between
time points. As expected, the percent
degradation increased with incubation
time, in correlation with a decreased
percent of the peak of interest as thesample degraded (Table 3). A narrowsmear analysis range of the peak ofinterest can help to identify varying
sizing from incomplete transcription. The
smear analysis feature offers a simple
approach for assessing sample quality.

Table 2. IVT RNA analyzed on the Agilent 5200 Fragment Analyzer system with the Agilent
RNA kit (15 nt) and separated with both the normal RNA method and the extended RNAmethod (A) 9,000 nt sample (B) 10,000 nt sample. *n=3.

RFUDegradation series

101412Size (bp)

16Long RNALadder

9,000
6,000
5,000
4,000
3,000
2,500
2,000
1,500
1,000500

15Figure 4. 9,000 nt IVT mRNA analyzed on the Agilent 5200 Fragment Analyzer system with the Agilent
RNA kit (15 nt) using the long RNA Ladder and extended RNA method over a degradation series at 70 °C
from 0 to 16 minutes. (A) Electropherogram overlay of degradation series. (B) Gel image of degradation.
LM = lower marker.

5Smear Analysis of Degradation Series 9,000 nt IVT RNA
70 °C Incubation
Time (minutes)

Percent of Degradation(75 to 7,500 nt)*

Percent of Peak of Interest(7,500 to 11,000 nt)*

036.8 %

62.7 %

244.6 %

54.9 %

452.3 %

46.7 %

660.8 %

36.5 %

870.0 %

28.1 %

1077.3 %20.9 %

1285.3 %13.4 %

1487.7 %10.4 %

1690.5 %7.5 %32,000Conclusionwww.agilent.com
For Research Use Only. Not for use in diagnostic procedures.
This information is subject to change without notice.
© Agilent Technologies, Inc. 2019
Printed in the USA, May 1, 20195994-0878EN

24,00020,000RFU

16,00012,000
8,000

9,000

0LM
1,500
2,000
2,500
3,000
4,000
5,000
6,000

4,000

1,000

The Agilent 5200 Fragment Analyzer
system offers reliable quality control
analysis essential to IVT mRNA
workflows. Accurate sizing of IVT mRNA
greater than 6,000 nt was achieved withthe Agilent RNA kit (15 nt) using a long
RNA Ladder with both the normal andextended RNA method. The extended
RNA method offers the option of
exceptional sizing to confirm complete
transcription and the ability to distinguishminute amounts of degradation. The
ProSize Smear Analysis tab in theAgilent ProSize data analysis software
enables the simultaneous detection of
the percent of the peak of interest andthe percent of degradation. This feature
aids the user in quickly identifying
a quality sample. In addition, the5200 Fragment Analyzer and the RNA kit(15 nt) provided accurate sizing with theaddition of a 7-methylguanylate cap at
the 5’ end of the RNA transcript.

28,000500

A common modification made to IVTmRNA transcript is the addition of a7-methylguanylate cap at the 5’ end.
A 5’ cap stabilizes the RNA transcript,prevents degradation, and assists inbinding during translation. Capped anduncapped 1,800 nt IVT mRNA wereanalyzed on the 5200 Fragment Analyzer
system with the RNA kit (15 nt) using
the extended RNA method and long RNA
Ladder (Figure 5). The extended RNA
method was used to provide the best
resolution possible. An average sizeof 1,810 nt was observed for both thecapped and uncapped RNA transcripts.
RNA capping did not influence the sizing
or general profile of the transcript.

Table 3. Smear analysis of the percent concentration for two regionsof the 9,000 nt IVT mRNA throughout the degradation series separated
on the Agilent 5200 Fragment Analyzer system with the Agilent RNA kit(15 nt) using the extended RNA method and the long RNA Ladder. *n=2.

15

Capped versus uncappedSize (nt)

Figure 5. Overlay of the 1,800 nt IVT mRNA fragments capped (red) and uncapped (black) separated
on the Agilent 5200 Fragment Analyzer system with the Agilent RNA kit (15 nt) using the extended RNAmethod and the long RNA Ladder. LM= lower marker

Reference1.

Warzak, D.; Pocernich, C.; Wong, K-S.
Benefits of Quality Control in the
IVT RNA Workflow Using theAgilent 5200 Fragment Analyzer
System, Agilent Technologies
Application Note, publication number
5994‑0512EN, 2018.