| RNAi
MicroRNA
Headquarters: North America:
Exiqon A/S Exiqon, Inc.
Bygstubben 9 14 F Gill Street
2950 Vedbaek Woburn, MA 01801, USA
Denmark Phone: +1 781 376 4150
Phone: +45 45 66 08 88 Toll free: +1 888 miRCURY
Fax: +45 45 66 18 88 Fax: +1 781 376 4152
Email: exiqon@exiqon.com Email: exiqon@exiqon.com
www.exiqon.com www.exiqon.com
MicroRNA Profi ling Using miRCURY
TM
LNA Array
This protocol is for a microRNA profi le using the miRCURY
TM
5. Flush hyb. chamber with 1x Hybridization buffer
LNA Arrays with the Tecan HS4800
TM
Pro Hybridization Check the appropriate volume of the chamber in the suppliers
Station. Protocols for manual hybridization are available at manual and add 1x diluted Hybridization buffer. Dilute with
www.exiqon.com. water.
Before starting the experiment:
6. Inject reaction mixture
Use the miRCURY
TM
LNA microRNA Power labeling kit for labeling of
Inject the 50 µL target preparation to the hybridization station.
your sample(s).
7. Incubate at 56° C for 16 h.
Dissolve the Spike-in miRNA in 30 µl of nuclease free water.
Set the program for the hybridization station to 56° C and 16 h.
Leave the suspension on ice for 30 minutes to dissolve. Vortex
incubation. Agitation should be set to medium, if possible.
and then spin to collect tube contents. Store the dissolved spike-in
miRNA at -20° C until use.
8. Two runs of wash at 56° C for 1 min. using Wash buffer A
Set the program for the hybridization station accordingly.
Please refer to the instruction manual of your hybridization station
for correct volume of buffers required to perform the hybridization.
9. Two runs of wash at 23° C for 1 min. using Wash buffer B
The volumes in Table 1 applies to the hybridization of 4 slides in
Set the program for the hybridization station accordingly.
a Tecan HS400/HS4800 hybridization station. Protocols for various
automated hybridization stations are available at www.exiqon.com.
10. Two runs of wash at 23° C for 1 min. using Wash buffer C
Set the program for the hybridization station accordingly.
Protocol:

11. Wash at 23° C for 30 sec. using Wash buffer C
1. Combine the labeled sample(s)
Set the program for the hybridization station accordingly.
The two samples from the Hy3TM and Hy5TM labeling reactions
are combined on ice. Total volume should be 25 µL.
12. Dry slides
Set the program for the hybridization station accordingly.
2. Add 25 µL Hybridization buffer
Check for precipitation (see p. 12) in the Hybridization buffer
Total handling time: 1 hour
before adding 25 µL to the labeled sample(s). Mix by vortexing
and spin briefl y.
3. Incubate at 95° C for 2 min.
During the incubation the target preparation should be protected
from light.
4. Incubate 2 min. on ice
Leave on ice for at least 2 min. and up to 15 min.
Briefl y spin the reaction after ice incubation.
Table 1
Protocol Guide ı 2008 View entire protocol online at www.biotechniques.com/protocol ı BioTechniques ı 65
Exiqon Protocol.indd 65 10/26/07 12:01:32 PM
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