BTN Protocol Guide-08

Protocol for
Protocol:

microRNA proﬁ ling
1. Combine the labeled sample(s)
The two samples from the Hy3™ and Hy5™ labeling reactions are
using miRCURY™
combined on ice. Total volume should be 25 µL.
LNA Array
2. Add 25 µL Hybridization buffer
Check for precipitation (see p. 12) in the Hybridization buffer
before adding 25 µL to the labeled sample(s). Mix by vortexing and
This protocol is for a microRNA proﬁ le using the miRCURY™ spin brieﬂ y.
LNA Arrays with the Tecan HS4800™ Pro Hybridization Station.
Protocols for manual hybridization are available at www.exiqon.com 3. Incubate at 95° C for 2 min.
During the incubation the target preparation should be protected
We’re pushing
Before starting the experiment from light.
Use the miRCURY™ LNA microRNA Power labeling kit for labeling
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the frontiers of
of your sample(s). 4. Incubate 2 min. on ice
Leave on ice for at least 2 min. and up to 15 min.
As illustrated below, miRCURY
TM
LNA microRNA Arrays Dissolve the Spike-in miRNA in 30 µl of nuclease free water. Brieﬂ y spin the reaction after ice incubation.
microRNA analysis,
produce very speciﬁ c signals. You can create an effective, Leave the suspension on ice for 30 minutes to dissolve. Vortex and
valid, and uniquely speciﬁ c microRNA proﬁ le with minimal then spin to collect tube contents. Store the dissolved spike-in 5. Flush hyb. chamber with 1x Hybridization buffer
so you can accelerate
cross-hybridization from just 30 ng total RNA. miRNA at –20° C until use. Check the appropriate volume of the chamber in the suppliers
manual and add 1x diluted Hybridization buffer. Dilute with water.
discoveries.
Figure 1 Please refer to the instruction manual of your hybridization sta-
tion for correct volume of buffers required to perform the hybridi- 6. Inject reaction mixture
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zation. The volumes in Table 1 applies to the hybridization of 4 Inject the 50 µL target preparation to the hybridization station.
We have just launched the next generation of our popular
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slides in a Tecan HS400/HS4800 hybridization station.
Locked Nucleic Acid-based arrays – the miRCURY
TM
LNA �� Protocols for various automated hybridization stations are avail- 7. Incubate at 56° C for 16 h.
microRNA Array. able at www.exiqon.com Set the program for the hybridization station to 56° C and 16 h.
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incubation. Agitation should be set to medium, if possible.
With more sensitivity and speciﬁ city than ever before, you
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Table 1
can conduct expression proﬁ ling of a comprehensive range 8. Two runs of wash at 56° C for 1 min. using Wash buffer A
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of microRNAs from any organism you like – vertebrates, Recipes for preparation of 200 mL Wash buffers Set the program for the hybridization station accordingly.
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invertebrates, plants and viruses – from a strikingly small
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amount of sample. You even have access to 150 proprietary
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Wash Wash Wash 9. Two runs of wash at 23° C for 1 min. using Wash buffer B
microRNAs unavailable elsewhere (miRPlus
TM
). � buffer A buffer B buffer C
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Set the program for the hybridization station accordingly.
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20x Salt buffer 20 mL 10 mL 2 mL
Our microRNA array platform is part of Exiqon’s
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10. Two runs of wash at 23° C for 1 min. using Wash buffer C
miRCURY
TM
product line, the most complete range of tools
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10% Detergent solution 4 mL - -
Set the program for the hybridization station accordingly.
for microRNA analysis available today. The miRCURY
TM
tools
Figure 1: The unique speciﬁ city of the miRCURY
TM
LNA microRNA Arrays
Nuclease-free water 176 mL 190 mL 198 mL
enable you to seek, ﬁ nd and verify microRNAs – and to
A total of 431 microRNAs were divided into eight pools with 40-71 distantly
11. Wash at 23° C for 30 sec. using Wash buffer C
accelerate your discoveries.
related microRNAs in each pool and analyzed using miRCURY
TM

Set the program for the hybridization station accordingly.
LNA microRNA Arrays. The resulting data demonstrate the high speciﬁ city
Dr. Carsten Alsbo, miRCURY
TM
Array Product Manager
of the miRCURY
TM
LNA microRNA Arrays and a maximum cross-
12. Dry slides
hybridization of just 4.9% across all eight pools, with an average of 3.9%.
Set the program for the hybridization station accordingly.
Learn more about research based on Exiqon’s miRCURY
TM
Total handling time: 1 hour
LNA microRNA arrays at www.exiqon.com/array.
Contact our research specialists to learn more
North America: +1 781 376 4150 • Rest of world: +45 45 650 929
support@exiqon.com • www.exiqon.com
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