75$16*(120,&
Optimized Purification of siRNA Oligonucleotides
Using the WAVE® Oligo System

Introduction
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APPLICATION NOTE 120

Synthetic siRNA oligonucleotides represent powerful tools for
RNAi-mediated gene silencing studies. Highly pure siRNAoligonucleotides can result in increased delivery efficiencies
and reduce the risk of toxic effects from contaminants, thereby
improving the success rates and reproducibility of gene silencing
experiments. Here we describe an optimized siRNA oligonucleotide
purification protocol using the OligoSep™ Prep HC Cartridge andWAVE Optimized® HA Buffers specifically developed for oligonucleotide
purification applications on the WAVE Oligo System.

Materials and Methods
Oligonucleotide Synthesis
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Post-column Preparation
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siRNA Oligonucleotide Purification)XOOOHQJWKSURGXFWZDVSXULILHGIURP
DSSUR[LPDWHO\2'VRIFUXGHPDWHULDO
RQ WKH :$9( 2OLJR 6\VWHP XVLQJ WKH
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Sequences of siRNA Oligonucleotides
StrandLamin A/C

Luciferase

Sense Strand

CUGGACUUCCAGAAGAACAdTdTCGUACGCGGAAUACUUCGAdTdT

Antisense Strand

UGUUCUUCUGGAAGUCCAGdTdTUCGAAGUAUUCCGCGUACGdTdT

Table 2
Purification Gradient Method
Step

Time (min)LoadingStart Gradient
Stop GradientStart Clean
Stop Clean
Start Equilibrate
Stop Equilibrate

0
0.5
16.5
16.6
17.6
17.7
17.8

Percent WAVE Optimized Percent WAVE Optimized
HA Buffer A
HA Buffer B
80
20
70
30
40
60
0
100
0
100
80
20
80
20

Column: OligoSep Prep HC Cartridge, 7.8 x 50 mm
WAVE Optimized HA Buffer A: 100 mM n-Hexylammonium Acetate/10% Acetonitrile
WAVE Optimized HA Buffer B: 100 mM n-Hexylammonium Acetate/50% Acetonitrile
Flow rate: 1.2 ml/min
Temperature: 80o C
Sample Loop: 500 µl
Sample Injection: 100 µl
Dead Time: 2.7 min
Detection: UV at 300 nm in preparative mode, 260 nm in analytical mode

Table 3
Percent Full-Length Material Following Purification on the WAVE Oligo System
siRNA Oligonucleotide
Lamin A/C Sense
Lamin A/C Antisense
Luciferase Sense
Luciferase AntisensePercent Full-Length Crude
36
50
36
48

Percent Full-Length Purified
>95
>95
>95
>95

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295

Results

245

Intensity (mV)

195

Synthesis Failuresand Impurities

145

95

45

-5
0

5

10

15

20

25

30

35

40

45

50

Time (min)Figure 1. Ion exchange HPLC analysis of crude siRNA oligonucleotide prior topurification (siRNA oligonucleotide: Luciferase Antisense).

500
450
400

Intensity (mV)

350
300
250

Full-Length Product

200

Synthesis Failuresand Impurities

150
100
50
0
0

2

4

6

8

10

12

14

16

18

20

Time (min)Figure 2. Example chromatogram using the optimized siRNA reverse-phase denaturing HPLC purification protocol described here for the WAVE Oligo System (siRNAoligonucleotide: Luciferase Antisense).

160
140

Luciferase Antisense

120

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Full-Length Product(50% of product)

100

Luciferase Sense
80
60

Lamin AC Antisense
40
20

Lamin AC Sense
0
0

2

4

6

8

10

12

14

16

18

20

Time (min)Figure 3. Analytical ion exchange HPLC analyses of siRNA oligonucleotides following
purification on the WAVE Oligo System.

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Application Note Product List
Product

Catalog Number

WAVE Oligo System

WAV-99-0004A

Oligo Sep Prep HC Cartridge
Oligo-MP Cartridge (10 pack)

NUC-99-3860
21-8560-86

Oligo-MP Cartridge (100 pack)

21-8560-90

WAVE Optimized HA Buffer A

553415

WAVE Optimized HA Buffer B

553416

T lcaa CPG Support

19-7640-00

www.transgenomic.com

Corporate Headquarters

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United Kingdom

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Soei Bldg. 4F
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Fax: (81) 3-5338-0184
E-mail: info@transgenomic.co.jp

WAVE, WAVE Optimized, TRANSGENOMIC, the globe logo and “transforming the world” are registered trademarks, andOligoSep is a trademark of Transgenomic, Inc. ©2003 Transgenomic, Inc. All rights reserved. Printed in USA.

Document No. 612021-00